Phosphorous protecting groups and methods of preparation and use thereof

ABSTRACT

Aspects of the present disclosure include compositions that make use of phosphorus and/or nucleobase protecting groups which find use in the synthesis of long polynucleotides. Phosphorus protecting groups are provided that help increase the stepwise coupling yield and/or phosphorous protecting groups that can be removed during the oxidation step. Amidine nucleobase protecting groups are provided that find use in the subject compositions and methods which provides for e.g., increased resistance to depurination during polynucleotide synthesis. In some instances, the methods and compositions disclosed herein utilize a combination of the phosphorus and amidine nucleobase protecting groups in the synthesis of polynucleotides having a sequence of 200 or more monomeric units in length. Also provided are methods for synthesizing a polynucleotide (e.g., a DNA) using one or more compounds disclosed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/173,143, filed Oct. 29, 2018 which is a divisionalapplication of U.S. patent application Ser. No. 14/701,288, filed Apr.30, 2015, issued as U.S. Pat. No. 10,196,418 on Feb. 5, 2019 whichclaims priority to U.S. provisional application Ser. No. 61/986,594,filed Apr. 30, 2014, the disclosure of these applications is hereinincorporated by reference.

The content of the ASCII text file of the sequence listing named13R6724.TXT, which is 1.088 kb in size was created on and electronicallysubmitted via EFS-Web on Jul. 19, 2021, is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to synthesis of nucleic acids. Moreparticularly, the invention relates to novel phosphorous protectinggroups and novel nucleobase protecting groups and related compoundsuseful in synthesis of DNAs, and compositions and methods thereof.

INTRODUCTION

Chemical synthesis of oligonucleotides, synthetic strands of DNA andRNA, is the chemical synthesis of relatively short fragments of nucleicacids with defined chemical structure and sequence. The technique isextremely important and useful owing to the wide variety of applicationsin current laboratory practice. Oligonucleotide synthesis provides arapid and inexpensive access to custom-made oligonucleotides of thedesired sequence. Oligonucleotides find a variety of applications inmolecular biology and medicine, e.g., antisense oligonucleotides, smallinterfering RNA, primers for DNA sequencing and amplification, molecularprobes, etc.

Chemical synthesis of oligonucleotide is typically carried out in 3′ to5′ direction as solid-phase synthesis using phosphoramidite method andphosphoramidite building blocks derived from protected2′-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, andU), or chemically modified nucleosides, e.g., 2-O-methyl, 2′-F, LNA,etc. To synthesize oligonucleotide, the building blocks are sequentiallycoupled to the growing oligonucleotide chain in the desired order. Oncethe chain assembly is complete, the product is deprotected, releasedfrom the solid phase to solution, and collected. (U.S. Pat. No.4,415,732; McBride, et al. 1983 Tetrahedron Letters 24:245-248; Sinha,et al. 1984 Nuc. Acids Res. 12:4539-4557.)

DNA synthesis with cyanoethyl phosphoramidites has been powerful atenabling synthesis of oligonucleotides of 200 mers or shorter on solidsupport and on arrays synthesis. However, the currently availablechemistry has encountered difficulties with the synthesis of longeroligonucleotides, which has been found to contain single base deletions(SBD) in their sequences and large deletions (deletions of longersegments of oligos). Single base deletions may occur due to insufficientstepwise coupling yields, whereas large deletions of oligonucleotidesegments can be due to the instability of the phosphotriester linkagesexposed to the repeated treatments of harsh chemicals during theoligonucleotide synthesis.

Thus, for existing synthetic methodologies, the undesired side reactionshave set practical limits for the length of synthetic oligonucleotides(up to about 200 nucleotide residues) because the number of errorsaccumulates with the length of the oligonucleotide being synthesized.(Beaucage, et al. 1992 “Advances in the Synthesis of Oligonucleotides bythe Phosphoramidite Approach” Tetrahedron 48 (12): 2223; see also,Caruthers, et al. 1987 “Synthesis of oligonucleotides using thephosphoramidite method” Biophosphates and their Analogues Synthesis,Structure, Metabolism and Activity, eds. K. S. Bruzik, W. J. Stec,Elsevier Sci. Publ., 3-21.) Since errors in the desired sequence aremore likely as the sequence length increases, sequences of more than 80bases (in particularly, more than 200 bases) often need to be isolatedby high-performance liquid chromatography (HPLC) to increase purity.Moreover, the stepwise yield or coupling efficiency greatly limits thelength of the oligonucleotide that can be synthesized. The overall yieldfor the synthesis of an oligonucleotide is expressed as OY=y^((n-1));with OY being the Overall Yield, y the stepwise coupling yield and n thelength of the oligonucleotide or the number of nucleotides in theoligonucleotide; thus a 200 mers oligonucleotide synthesized with a99.7% coupling yield will have a OY=y¹⁹⁹, which equals toOY=0.997¹⁹⁹=0.54996 or 54.99% whereas a 200 mers synthesized with a99.8% coupling efficiency will have a OY=0.671394 or 67.14%. Thestepwise coupling yield affects drastically the Overall Yield for suchlong oligonucleotides, as demonstrated in this example where a 0.1%difference in the stepwise coupling yield results in a 12% change in OY.As a result, any improvements on the stepwise coupling efficiency willgreatly increase the OY of the full length oligonucleotide and isdesired.

A significant challenge remains in chemical synthesis ofoligonucleotide. In particular, new phosphorus protecting groups areneeded that increase the stepwise coupling yield and/or phosphorousprotecting groups that can be removed during the oxidation step so as toavoid the instability of the phosphotriester linkage during theoligonucleotide synthesis as discussed previously. This is a key unmetneed for novel approaches of reliable synthesis of nucleic acidmolecules of greater length than those produced by conventionaltechniques, while achieving acceptable purity and yield.

SUMMARY

The present disclosure is based in part on the novel approaches tosynthesize nucleic acid molecules of greater length than those producedby conventional techniques, while achieving acceptable purity and yield.Aspects of the present disclosure are methods and compositions that makeuse of phosphorus and/or nucleobase protecting groups that provide forthe synthesis of long polynucleotides (e.g., DNA) having a sequence of200 or more monomeric units in length.

Phosphorus protecting groups are provided that help increase thestepwise coupling yield and/or phosphorous protecting groups that can beremoved during the oxidation step. Amidine nucleobase protecting groupsare provided that find use in the subject compositions and methods whichprovides for increased resistance to depurination during polynucleotidesynthesis, reduced nucleobase deprotection times, e.g., duringpolynucleotide cleavage in ammonia, and increased purity of crudeoligonucleotides with less byproducts, e.g., nucleobase adducts. In someinstances, the methods and compositions disclosed herein may utilize acombination of the phosphorus and nucleobase protecting groups in thesynthesis of polynucleotides having a sequence of 200 or more monomericunits in length.

In one aspect, the present disclosure generally relates to a compoundhaving the structural formula (I):

wherein B is a nucleobase or an analogue thereof, each of R₁ and R₂ isindependently a linear, branched or cyclic, substituted orun-substituted alkyl, or R₁ and R₂ together form a 5-, 6-, 7- or8-membered non-aromatic ring; R₃ is an acid-labile protecting group; andR is a group selected from the group consisting of benzyl alcoholderivatives, alpha-methyl aryl alcohols derivatives, naphthalene alcoholderivatives, bi-cyclic aliphatic alcohol derivatives or fused rings),S-ethylthioate derivatives and amino acid derivatives, with the provisothat R is not o-methyl benzyl.

In another aspect, the present disclosure generally relates to a methodfor synthesizing a polynucleotide (e.g., a DNA) using one or morecompounds disclosed herein, wherein the synthesized DNA is of a lengthof at least about 200 nucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows TIC and HPLC chromatograms and FIG. 1 (cont'd) shows Massanalysis of the synthesis of dT₂₀ using 1-(4-Bromophenyl)ethanol asphosphorus protecting group.

FIG. 2 shows TIC and HPLC chromatograms and FIG. 2 (cont'd) shows Massanalysis of the synthesis of dT₂₀ using 1-methoxy-2-naphtalenemethanolas phosphorus protecting group.

FIG. 3 shows TIC and FIG. 3 (cont'd) shows HPLC chromatograms, FIG. 3(cont'd 2) shows a portion of HPLC chromatograms of FIG. 3 (cont'd) andFIG. 3 (cont'd 3) shows Mass analysis of the synthesis of dT₂₀ using2-naphtalenemethanol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment.

FIG. 4 shows TIC and FIG. 4 (cont'd) shows IPLC chromatograms and FIG. 4(cont'd 2) shows Mass analysis of the synthesis of dT₂₀ using6-Bromo-2-naphtalenemethanol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment.

FIG. 5 shows TIC and FIG. 5 (cont'd) shows HPLC chromatograms and FIG. 5(cont'd 2) shows Mass analysis of the synthesis of dT₂₀ using1-hydroxyindane-5-carbonitrile as phosphorus protecting group.

FIG. 6 shows TIC and FIG. 6 (cont'd) shows HPLC chromatograms and FIG. 6(cont'd 2) shows Mass analysis of the synthesis of dT₂₀ using4-cyanobenzyl alcohol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment.

FIG. 7 shows TIC and FIG. 7 (cont'd) shows HPLC chromatograms and FIG. 7(cont'd 2) shows Mass analysis of the synthesis of dT₂₀ using3-cyanobenzyl alcohol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment.

FIG. 8 shows TIC and FIG. 8 (cont'd) shows HPLC chromatograms and FIG. 8(cont'd 2) shows Mass analysis of the synthesis of dT₂₀ using2-ethynylbenzyl alcohol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment.

FIG. 9 shows TIC and FIG. 9 (cont'd) shows IPLC chromatograms and FIG. 9(cont'd 2), FIG. 9 (cont'd 3) and FIG. 9 (cont'd 4) show Mass analysisof the synthesis of dT₂₀ using Acetyl-L-threoninemethylester asphosphorus protecting group.

FIG. 10 shows TIC and HPLC chromatograms and FIG. 10 (cont'd) shows Massanalysis of the synthesis of dT₂₀ using S-ethylbenzothioate asphosphorus protecting group.

FIG. 11 shows TIC and IPLC chromatograms and FIG. 11 (cont'd) and FIG.11 (cont'd 2) show Mass analysis of the synthesis of dT₂₀ using4-cyanobenzyl alcohol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment.

FIG. 12 shows TIC and HPLC chromatograms and FIG. 12 (cont'd) and FIG.12 (cont'd 2) show Mass analysis of the synthesis of dT₆₀ usingAcetyl-L-threoninemethylester as phosphorus protecting group.

FIG. 13 shows TIC and HPLC chromatograms and FIG. 13 (cont'd) and FIG.13 (cont'd 2) show Mass analysis of the synthesis of dT₆₀ usingS-ethylbenzothioate as phosphorus protecting group.

FIG. 14 shows a comparison of TIC and HPLC chromatograms and FIG. 14(cont'd) shows Mass analysis of a T₆₀ synthesized usingS-ethylbenzothioate as phosphorus protecting group and a T₆₀ synthesizedwith a standard 2-cyanoethyl phosphoramidite.

FIG. 15 and FIG. 15 (cont'd) depict the structures of phosphoramiditecompounds of interest (22-31) used in the synthesis of theoligonucleotides selected and shown in FIG. 1 to FIG. 14.

FIG. 16 shows the IPLC chromatogram of a d(AAT)₂₀ 60mers oligonucleotidesynthesized with standard benzoyl protecting group on the amino N-6 ofadenosine.

FIG. 17 and FIG. 17 (cont'd) show two IPLC chromatograms of a d(AAT)₂₀60mers oligonucleotide synthesized with respectivelyN⁶—(N,N-dimethylamidino) protecting group on the amino N-6 of adenosine(top) and N⁶-(1-(morpholino)ethylidene)-dA (bottom).

FIG. 18 and FIG. 18 (cont'd) show two IPLC chromatograms of a (CCT)₁₉C₃60mers oligonucleotide synthesized with respectively N⁴acetyl dCstandard protecting group (top) and N⁴—(N-methyl-2-pyrrolidinyldene)-dC(bottom).

FIG. 19 shows a IPLC chromatogram of a dC₄₀ synthesized withN⁴-(1-(morpholino) ethylidene)-dC.

FIG. 20 shows a IPLC chromatogram of a d(GGT)₂₀ a 60mers oligonucleotidesynthesized with standard N²-(isobutyryl)-dG.

FIG. 21 and FIG. 21 (cont'd) show two IPLC chromatograms of a d(GGT)₂₀60mers oligonucleotide synthesized with respectivelyN²—(N,N-dimethylamidino) dG (top) and N²-(1-(morpholino)ethylidene)-dG(bottom).

FIG. 22 shows comparative 4 TIC chromatograms of 60mers oligonucleotidessynthesized with standard protecting groups and amidine protectinggroups. The decrease of base adducts is noticeable when the amidineprotecting groups are used.

FIG. 23 shows a IPLC chromatogram of a d(AAT)₁₃ 39mers oligonucleotidesynthesized with N⁶—(N,N-dimethylamidino)-dA-3′O—S-(ethyl)benzothioatephosphoramidite.

FIG. 24 and FIG. 24 (cont'd) show two comparative HPLC chromatograms oftwo 100mers oligonucleotides (SEQ ID NO: 2) synthesized respectivelywith standard nucleobase protecting groups (FIG. 24) and amidineprotecting groups (FIG. 24 (cont'd).

FIG. 25 depicts a denaturing polyacrylamide gel electrophoresis of a200mer polynucleotide synthesized on an array using3′-S-(ethyl)benzothioate phosphoramidites, and N⁶—(N,N-dimethylamidino)dA (compounds 16, 17, 18, 20).

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. General principles of organic chemistry,as well as specific functional moieties and reactivity, are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999.

Certain compounds of the present disclosure may exist in particulargeometric or stereoisomeric forms. The present disclosure contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the present disclosure. Additional asymmetric carbon atoms maybe present in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this presentdisclosure.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present disclosure. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios arecontemplated by the present disclosure. Those of ordinary skill in theart will readily appreciate that analogous ratios are contemplated formore complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentdisclosure is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic methods well known in the art, and subsequent recoveryof the pure enantiomers.

Given the benefit of this disclosure, one of ordinary skill in the artwill appreciate that synthetic methods, as described herein, may utilizea variety of protecting groups. By the term “protecting group”, as usedherein, it is meant that a particular functional moiety, e.g., O, S, orN, is temporarily blocked so that a reaction can be carried outselectively at another reactive site in a multifunctional compound. Inpreferred embodiments, a protecting group reacts selectively in goodyield to give a protected substrate that is stable to the projectedreactions; the protecting group should be selectively removable in goodyield by preferably readily available, non-toxic reagents that do notattack the other functional groups; the protecting group forms an easilyseparable derivative (more preferably without the generation of newstereogenic centers); and the protecting group has a minimum ofadditional functionality to avoid further sites of reaction. Oxygen,sulfur, nitrogen, and carbon protecting groups may be utilized. Examplesof a variety of protecting groups can be found in Protective Groups inOrganic Synthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., JohnWiley & Sons, New York: 1999.

Terms and symbols of nucleic acid chemistry, biochemistry, genetics, andmolecular biology used herein follow those of standard treatises andtexts in the field, e.g. Kornberg and Baker, DNA Replication, SecondEdition (W.H. Freeman, New York, 1992); Lehninger, Biochemistry, SecondEdition (Worth Publishers, New York, 1975); Strachan and Read, HumanMolecular Genetics, Second Edition (Wiley-Liss, New York, 1999);Eckstein, editor, Oligonucleotides and Analogs: A Practical Approach(Oxford University Press, New York, 1991); Gait, editor, OligonucleotideSynthesis: A Practical Approach (IRL Press, Oxford, 1984); Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold SpringHarbor Laboratory, 1989); and the like. Still, certain terms are definedbelow for the sake of clarity and ease of reference.

It will be appreciated that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties.

The terms “nucleotide” or “nucleotide moiety”, as used herein, refer toa sub-unit of a nucleic acid (whether DNA or RNA or analogue thereof),which includes a phosphate group, a sugar group and a heterocyclic base,as well as analogs of such sub-units. Other groups (e.g., protectinggroups) can be attached to any component(s) of a nucleotide.

The terms “nucleoside” or “nucleoside moiety”, as used herein, refer anucleic acid subunit including a sugar group and a heterocyclic base, aswell as analogs of such sub-units. Other groups (e.g., protectinggroups) can be attached to any component(s) of a nucleoside.

The terms “nucleoside” and “nucleotide” are intended to include thosemoieties that contain not only the known purine and pyrimidine bases,e.g. adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U),but also other heterocyclic bases that have been modified. Suchmodifications include methylated purines or pyrimidines, alkylatedpurines or pyrimidines, acylated purines or pyrimidines, halogenatedpurines or pyrimidines, deazapurines, alkylated riboses or otherheterocycles. Such modifications include, e.g., diaminopurine and itsderivatives, inosine and its derivatives, alkylated purines orpyrimidines, acylated purines or pyrimidines, thiolated purines orpyrimidines, and the like, or the addition of a protecting group such asacetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl,9-fluorenylmethoxycarbonyl, phenoxyacetyl, and substitutedphenoxyacetyl, dimethylformamidine, dibutylformamidine,pyrrolodinoamidine, morpholinoamidine, and other amidine derivatives,N,N-diphenyl carbamate, or the like. The purine or pyrimidine base mayalso be an analog of the foregoing; suitable analogs will be known tothose skilled in the art and are described in the pertinent texts andliterature. Common analogs include, but are not limited to,7-deazaadenine, 1-methyladenine, 2-methyladenine, N6-methyladenine,N6-isopentyladenine, 2-methylthio-N6-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and2,6-diaminopurine.

A “nucleobase” references the heterocyclic base of a nucleoside ornucleotide.

In addition, the terms “nucleoside” and “nucleotide” include thosemoieties that contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups including locked nucleic acids known as LNA and UNA unlockednucleic acids, 2′-fluoro, 2′-O-alkyl, 2′-O-ethoxymethoxy, or arefunctionalized as ethers, amines, or the like.

The term “analogues”, as used herein, refer to molecules havingstructural features that are recognized in the literature as beingmimetics, derivatives, having analogous structures, or other like terms,and include, for example, polynucleotides incorporating non-natural (notusually occurring in nature) nucleotides, unnatural nucleotide mimeticssuch as 2′-modified nucleosides, peptide nucleic acids, oligomericnucleoside phosphonates, and any polynucleotide that has addedsubstituent groups, such as protecting groups or linking groups.

The term “nucleic acid”, as used herein, refers to a polymer of anylength, e.g., greater than about 2 bases, greater than about 10 bases,greater than about 100 bases, greater than about 500 bases, greater than1,000 bases, up to about 10,000 or more bases composed of nucleotides,e.g., deoxyribonucleotides or ribonucleotides, and may be producedenzymatically or synthetically (e.g., PNA as described in U.S. Pat. No.5,948,902 and the references cited therein) which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleotides, e.g., canparticipate in Watson-Crick base pairing interactions.Naturally-occurring nucleotides include guanosine and 2′-deoxyguanosine,cytidine and 2′-deoxycytidine, adenosine and 2′-deoxyadenosine,thymidine and uridine (G, dG, C, dC, A, dA and T, U respectively).

A nucleic acid may exist in a single stranded or a double-stranded form.A double stranded nucleic acid has two complementary strands of nucleicacid may be referred to herein as the “first” and “second” strands orsome other arbitrary designation. The first and second strands aredistinct molecules, and the assignment of a strand as being a first orsecond strand is arbitrary and does not imply any particularorientation, function or structure. The nucleotide sequences of thefirst strand of several exemplary mammalian chromosomal regions (e.g.,BACs, assemblies, chromosomes, etc.), as well as many pathogens, areknown, and may be found in NCBI's Genbank database, for example. Thesecond strand of a region is complementary to that region.

The term “oligonucleotide”, as used herein, refers to a single strandedmultimer of nucleotides of, inter alia, from about 2 to 500 nucleotides.Oligonucleotides may be synthetic or may be made enzymatically, and, insome embodiments, are 10 to 50 nucleotides in length. Oligonucleotidesmay contain ribonucleotide monomers (i.e., may be oligoribonucleotides)or deoxyribonucleotide monomers. Oligonucleotides may contain, interalia, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51-60, 61 to 70, 71 to 80,80 to 100, 100 to 150, 150 to 500 or greater than 500 nucleotides inlength, for example.

The terms “deoxyribonucleic acid” and “DNA”, as used herein, refers to anucleic acid composed of nucleotides and/or deoxyribonucleotides.

The terms “ribonucleic acid” and “RNA”, as used herein, refer to anucleic acid composed of nucleotides and/or ribonucleotides.

An “internucleotide bond” or “nucleotide bond” refers to a chemicallinkage between two nucleoside moieties, such as the phosphodiesterlinkage in nucleic acids found in nature, or linkages well known fromthe art of synthesis of nucleic acids and nucleic acid analogues. Aninternucleotide bond may include a phospho or phosphite group, and mayinclude linkages where one or more oxygen atoms of the phospho orphosphite group are either modified with a substituent or a protectinggroup or replaced with another atom, e.g., a sulfur atom, or thenitrogen atom of a mono- or di-alkyl amino group.

The terms “heterocycle”, “heterocyclic”, “heterocyclic group” or“heterocyclo”, as used herein, refer to fully saturated or partially orcompletely unsaturated cyclic groups having at least one heteroatom inat least one carbon atom-containing ring, including aromatic(“heteroaryl”) or nonaromatic (for example, 3 to 13 member monocyclic, 7to 17 member bicyclic, or 10 to 20 member tricyclic ring systems). Eachring of the heterocyclic group containing a heteroatom may have 1, 2, 3,or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/orsulfur atoms, where the nitrogen and sulfur heteroatoms may optionallybe oxidized and the nitrogen heteroatoms may optionally be quaternized.The heterocyclic group may be attached at any heteroatom or carbon atomof the ring or ring system. The rings of multi-ring heterocycles may befused, bridged and/or joined through one or more spiro unions.Nitrogen-containing bases are examples of heterocycles. Other examplesinclude piperidinyl, morpholinyl and pyrrolidinyl.

The term “electron-withdrawing group” refers to a moiety that has atendency to attract valence electrons from neighboring atoms (i.e., thesubstituent is electronegative with respect to neighboring atoms). Aquantification of the level of electron-withdrawing capability is givenby the Hammett sigma constant. This well known constant is described inmany references, for instance, March, Advanced Organic Chemistry 251-59,McGraw Hill Book Company, New York, (1977). Electron-withdrawing groupsinclude nitro, acyl, formyl, sulfonyl, trifluoromethyl, cyano, chloride,and the like.

The term “electron-donating group” refers to a moiety that has atendency to repel valence electrons from neighboring atoms (i.e., thesubstituent is less electronegative with respect to neighboring atoms).Electron-donating groups include amino, methoxy, alkyl (including C1-6alkyl that can have a linear or branched structure), C4-9 cycloalkyl,and the like.

The phrase “protecting group”, as used herein, refers to a species whichprevents a portion of a molecule from undergoing a specific chemicalreaction, but which is removable from the molecule following completionof that reaction. A “protecting group” is used in the conventionalchemical sense as a group which reversibly renders unreactive afunctional group under certain conditions of a desired reaction, astaught, for example, in Greene, et al., “Protective Groups in OrganicSynthesis,” John Wiley and Sons, Second Edition, 1991. After the desiredreaction, protecting groups may be removed to deprotect the protectedfunctional group. All protecting groups should be removable (and hence,labile) under conditions which do not degrade a substantial proportionof the molecules being synthesized. In contrast to a protecting group, a“capping group” permanently binds to a segment of a molecule to preventany further chemical transformation of that segment. It should be notedthat the functionality protected by the protecting group may or may notbe a part of what is referred to as the protecting group.

The terms “hydroxyl protecting group” or “O-protecting group”, as usedherein, refers to a protecting group where the protected group is ahydroxyl. A “reactive-site hydroxyl” is the terminal 5′-hydroxyl during3′-5′ polynucleotide synthesis, or the 3′-hydroxyl during 5′-3′polynucleotide synthesis. A “free reactive-site hydroxyl” is areactive-site hydroxyl that is available to react to form aninternucleotide bond (e.g., with a phosphoramidite functional group)during polynucleotide synthesis.

The term “alkyl”, as used herein, refers to a saturated straight chain,branched or cyclic hydrocarbon group (e.g., having 1 to 24, typically 1to 12) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl,hexyl, isohexyl, cyclohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. Alkyls include “cycloalkyls”, which refer to cyclicalkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

The term “alkenyl”, as used herein, unless otherwise specified, refersto a branched, unbranched or cyclic (e.g. in the case of C₅ and C₆)hydrocarbon group of 2 to 24, typically 2 to 12, carbon atoms containingat least one double bond, such as ethenyl, vinyl, allyl, octenyl,decenyl, and the like. The term “lower alkenyl” intends an alkenyl groupof two to eight carbon atoms, and specifically includes vinyl and allyl.The term “cycloalkenyl” refers to cyclic alkenyl groups.

The term “alkynyl”, as used herein, unless otherwise specified, refersto a branched or unbranched hydrocarbon group of 2 to 24, typically 2 to12, carbon atoms containing at least one triple bond, such asacetylenyl, ethynyl, n-propynyl, isopropynyl, n-butynyl, isobutynyl,t-butynyl, octynyl, decynyl and the like. The term “lower alkynyl”intends an alkynyl group of two to eight carbon atoms, and includes, forexample, acetylenyl and propynyl, and the term “cycloalkynyl” refers tocyclic alkynyl groups.

The term “hydrocarbyl”, as used herein, refers to alkyl, alkenyl oralkynyl. Unless indicated otherwise, the term hydrocarbyl generallyencompasses “substituted hydrocarbyl”, which refers to hydrocarbylmoieties having substituents replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. Such substituents may include, for example,a hydroxyl, a halogen, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphonate, a phosphinate, an amino, an amido, an amidine, an imine, acyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, asulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclic, anaralkyl, or an aromatic or heteroaromatic moiety. It will be understoodby those skilled in the art that the moieties substituted on thehydrocarbon chain may themselves be substituted, if appropriate. Forinstance, the substituents of a substituted alkyl may includesubstituted and unsubstituted forms of amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, aswell as ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CN, and the like. Cycloalkyls may be furthersubstituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls,carbonyl-substituted alkyls, —CN, and the like.

The term “alkoxy” or “alkyloxy” means an alkyl group linked to oxygenand may be represented by the formula: R—O—, wherein R represents thealkyl group. An example is the methoxy group CH₃O—.

The term “aryl” refers to 5-, 6-, and 7-membered single- or multi-ringaromatic groups that may include, inter alia, from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings (the rings are “fused rings”) wherein at leastone of the rings is aromatic (e.g., the other cyclic rings may becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, and/or heterocycles).An example of a fused ring aryl group is naphthalene. A “lower aryl”contains up to 18 carbons, such as up to 14, 12, 10, 8 or 6 carbons.

The aromatic rings may be substituted at one or more ring positions withsuch substituents as described above for substituted hydrocarbyls, forexample, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclic, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like.

The term “halogen”, as used herein, refers to fluorine, chlorine,bromine, or iodine.

“Linkage” as used herein refers to a first moiety bonded to two othermoieties, wherein the two other moieties are linked via the firstmoiety. Typical linkages include ether (—O—), oxo (—C(O)—), amino(—NH—), amido (—N—C(O)—), thio (—S—), phospho (—P—), ester (—O—C(O)—).

The term “functionalized”, as used herein, refers to a process whereby amaterial is modified to have a specific moiety bound to the material,e.g., a molecule or substrate is modified to have the specific moiety;the material (e.g. molecule or support) that has been so modified isreferred to as a functionalized material (e.g., functionalized moleculeor functionalized support).

The term “substituted” as used to describe chemical structures, groups,or moieties, refers to the structure, group, or moiety comprising one ormore substituents. As used herein, in cases in which a first group is“substituted with” a second group, the second group is attached to thefirst group whereby a moiety of the first group (typically a hydrogen)is replaced by the second group. For example, when an alkyl group has a“R” group and R is hydrogen, the alkyl group is considered unsubstitutedat the marked location, whereas when that hydrogen is replaced with ahalogen, it is considered substituted by a halogen at that location.

The term “substituent”, as used herein, refers to a group that replacesanother group in a chemical structure. Typical substituents includenonhydrogen atoms (e.g. halogens), functional groups (such as, but notlimited to amino, sulfhydryl, carbonyl, hydroxyl, alkoxy, carboxyl,silyl, silyloxy, phosphate and the like), hydrocarbyl groups, andhydrocarbyl groups substituted with one or more heteroatoms. Exemplarysubstituents include alkyl, lower alkyl, aryl, aralkyl, lower alkoxy,thioalkyl, hydroxyl, thio, mercapto, amino, imino, halo, cyano, nitro,nitroso, azide, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl,silyloxy, boronyl, and modified lower alkyl.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.For example, the phrase “optionally substituted” means that anon-hydrogen substituent may or may not be present, and, thus, thedescription includes structures wherein a non-hydrogen substituent ispresent and structures wherein a non-hydrogen substituent is notpresent. At various points herein, a moiety may be described as beingpresent zero or more times: this is equivalent to the moiety beingoptional and includes embodiments in which the moiety is present andembodiments in which the moiety is not present. If the optional moietyis not present (is present in the structure zero times), adjacent groupsdescribed as linked by the optional moiety are linked to each otherdirectly. Similarly, a moiety may be described as being either (1) agroup linking two adjacent groups, or (2) a bond linking the twoadjacent groups. The descriptions (1) and (2) are equivalent to themoiety being optional and includes embodiments in which the moiety ispresent and embodiments in which the moiety is not present. If theoptional moiety is not present (is present in the structure zero times),adjacent groups described as linked by the optional moiety are linked toeach other directly.

“Isolated” or “purified” generally refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptide,chromosome, etc.) such that the substance comprises a substantialportion of the sample in which it resides (excluding solvents), i.e.greater than the substance is typically found in its natural orun-isolated state. Typically, a substantial portion of the samplecomprises at least about 1%, at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 50%, preferably atleast about 80%, or more preferably at least about 90% of the sample(excluding solvents). For example, a sample of isolated RNA willtypically comprise at least about 5% total RNA, where percent iscalculated in this context as mass (e.g. in micrograms) of total RNA inthe sample divided by mass (e.g. in micrograms) of the sum of (totalRNA+other constituents in the sample (excluding solvent)). Techniquesfor purifying polynucleotides and polypeptides of interest are wellknown in the art and include, for example, gel electrophoresis,ion-exchange chromatography, affinity chromatography, flow sorting, andsedimentation according to density. In some embodiments, one or more ofthe nucleotide composition(s) is in isolated form.

As used herein, (Cr-Cy) refers in general to groups that have from x toy (inclusive) carbon atoms. Therefore, for example, C₁-C₆ refers togroups that have 1, 2, 3, 4, 5, or 6 carbon atoms, which encompassC₁-C₂, C₁-C₃, C₁-C₄, C₁-C₅, C₂-C₃, C₂-C₄, C₂-C₅, C₂-C₆, and all likecombinations. (C₁-C_(2M)) and the likes similarly encompass the variouscombinations between 1 and 20 (inclusive) carbon atoms, such as (C₁-C₆),(C₁-C₁₂) and (C₃-C₁₂).

The terms “covalent” or “covalently”, as used herein, refer to thenature of a chemical bonding interaction between atoms. A covalent bondis a chemical bonding that involves the sharing of electron pairsbetween atoms. The stable balance of attractive and repulsive forcesbetween atoms when they share electrons is referred to as covalentbonding. The sharing of electrons allows each atom to attain theequivalent of a full outer shell, corresponding to a stable electronicconfiguration. Covalent bonding includes various kinds of interactions,e.g., σ-bonding, π-bonding, metal-to-metal bonding, agnosticinteractions, and three-center two-electron bonds.

The terms “non-covalent” or “non-covalently”, as used herein, refer tothe nature of a chemical bonding interaction between atoms. Anon-covalent bond is a type of chemical bonding that does not involvethe sharing of pairs of electrons, but rather involves more dispersedvariations of electromagnetic interactions. There are four commonlymentioned types of non-covalent interactions: hydrogen bonds, ionicbonds, van der Waals forces, and hydrophobic interactions.

The terms “purified” or “to purify”, as used herein, refer to theremoval of components (e.g., contaminants) from a sample.

DETAILED DESCRIPTION

The present disclosure provides novel approaches to reliable synthesisof nucleic acid molecules of greater length than those produced byconventional techniques, while achieving acceptable purity and yield.Aspects of the present disclosure are methods and compositions that makeuse of phosphorus and/or nucleobase protecting groups that provide forthe synthesis of long polynucleotides (e.g., DNA) having a sequence of200 or more monomeric units in length.

Phosphorus protecting groups are provided that help increase thestepwise coupling yield and/or phosphorous protecting groups that can beremoved during the oxidation step. Amidine nucleobase protecting groupsare provided that find use in the subject compositions and methods whichprovides for increased resistance to depurination during polynucleotidesynthesis, reduced nucleobase deprotection times, e.g., duringpolynucleotide cleavage in ammonia, and increased purity of crudeoligonucleotides with less byproducts, e.g., nucleobase adducts. In someinstances, the methods and compositions disclosed herein may utilize acombination of the phosphorus and nucleobase protecting groups in thesynthesis of polynucleotides having a sequence of 200 or more monomericunits in length.

Aspects of the present disclosure include new phosphorus protectinggroups that can be removed during iodine oxidation such that theinternucleotide bond is less susceptible to hydrolysis reactions (seee.g., the schematic below) that occur during the next oxidation cycles.This novel approach leads to oligonucleotides with fewer largedeletions.

Using the protecting groups of the present disclosure on theinternucleotide bond also offers the advantage of increased couplingefficiency of the phosphoramidite. The phosphorus protective group mostoften used for solid-phase phosphoramidite DNA synthesis is thecyanoethyl protective group. (Letsinger, et al. 1969 J. Am. Chem. Soc.91(12), 3360-5). This protecting group is removed via a beta-eliminationreaction under the same conditions that the heterobase protecting groupsare removed at the end of the synthesis using ammonia or an alkyl amine,as depicted in the following scheme:

A further aspect of the present disclosure is the design of a phosphorusprotecting group that minimizes the hydrolysis of the phosphotriesterinternucleotide bond that may occur in the practice of the currentcyanoethyl phosphoramidite chemistry. Instead, during the iodineoxidation, the phosphotriester internucleotide intermediate isconverted—by cleavage of the protecting group- to a phosphodiesterinternucleotide linkage that is stable to hydrolysis. The cleavage ofthe phosphorus protecting group can be incomplete, for example as muchas 50% but can still have a significant effect on preventing hydrolysisof the internucleotide bond. It is preferred that the cleavage isgreater than 50% and more preferred that it is close to 100%. A furtheraspect of this present disclosure is a protecting group that is cleavedat 50% to 100% during the iodine oxidation and then completely cleavedat the end of the oligonucleotide synthesis by nucleophilic attack usingfor example a thiolate reagent or derivative thereof or bybeta-elimination or alpha fragmentation using a base or basic amines ora combination.

Another aspect of the present disclosure is the addition of a base tothe iodine oxidation solution to facilitate and increase the cleavage ofthe phosphorus protecting group during the oxidation step. Examples ofsuch bases include t-butyl amine, diisopropyl amine, diethyl amine,triethyl amine, diisopropylethyl amine, DBU and other non-nucleophilicbases.

In one aspect, the present disclosure generally relates to a compoundhaving the structural formula (I):

wherein

B is a nucleobase or an analogue thereof;

each of R₁ and R₂ is independently a linear, branched or cyclic,substituted or un-substituted alkyl, or R₁ and R₂ together form a 5-,6-, 7- or 8-membered non-aromatic ring;

R₃ is an acid-labile protecting group; and

R is a phosphorus protecting group selected from the group consisting ofbenzyl alcohol derivatives (except o-methyl benzyl), alpha-methyl arylalcohols derivatives, naphthalene alcohol derivatives, bi-cyclicaliphatic alcohol derivatives, acylthioalkyl alcohol derivatives,S-ethylthioate derivatives and amino acid derivatives.

In some embodiments, B is a nucleobase or a protected nucleobase, wherethe nucleobase is selected from Adenine, Guanine, Thymine, Cytosine andUracil, or a derivative or analog thereof.

Any convenient protecting groups may be utilized in the subjectcompounds. In some embodiments, B is a protected nucleobase. In certainembodiments, the nucleobase may be a conventional purine or pyrimidinebase, e.g., adenine (A), thymine (T), cytosine (C), guanine (G) oruracil (U), or a protected form thereof, e.g., wherein the base isprotected with a protecting group such as acetyl, difluoroacetyl,trifluoroacetyl, isobutyryl, benzoyl, phenoxyacteyl,4-(t-butyl)phenoxyacetyl and the like. In certain embodiments, thenucleobase includes an amidine protecting group (e.g., as describedherein).

The purine or pyrimidine base may also be an analog of the foregoing;suitable analogs include, but are not limited to: 1-methyladenine,2-methyladenine, N⁶-methyladenine, N⁶-isopentyladenine,2-methylthio-N⁶-isopentyladenine, N,N-dimethyladenine, 8-bromoadenine,2-thiocytosine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine,4-acetylcytosine, 1-methylguanine, 2-methylguanine, 7-methylguanine,2,2-dimethylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine,8-methylguanine, 8-thioguanine, 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, 5-ethyluracil, 5-propyluracil,5-methoxyuracil, 5-hydroxymethyluracil, 5-(carboxyhydroxymethyl)uracil,5-(methylaminomethyl)uracil, 5-(carboxymethylaminomethyl)-uracil,2-thiouracil, 5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil,uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester,pseudouracil, 1-methylpseudouracil, queosine, inosine, 1-methylinosine,hypoxanthine, xanthine, 2-aminopurine, 6-hydroxyaminopurine,6-thiopurine and 2,6-diaminopurine.

R₁ and R₂ can be the same or different. R₁ and R₂ can be both linear,both branched or cyclic, or mixed alkyl groups. R₁ and R₂ can be bothun-substituted alkyls, or one of them is substituted, or both aresubstituted.

In some cases, R₁ and R₂ may be components of and together form a 5-,6-, 7- or 8-membered ring structure (ring Q as shown below), forexample, a non-aromatic ring.

In certain embodiments, each of R₁ and R₂ independently is a linear,branched or cyclic, substituted or un-substituted C₁-C₁₈ alkyl. Incertain embodiments, each of R₁ and R₂ independently is a linear orbranched un-substituted C₁-C₆ alkyl. In certain embodiments, each of R₁and R₂ independently is a linear C₁-C₃ alkyl (i.e., methyl, ethyl andpropyl). In certain embodiments, each of R₁ and R₂ independently is abranched C₃-C₆ alkyl. For example, in the compound of formula (I_(a)),each of R₁ and R₂ may be isopropyl, as shown in formula (I_(a)), orisobutyl.

In certain embodiments, ring Q is a 5- or 6-membered non-aromatic ring,wherein the ring has 0 or 1 hetero-atom in the backbone. In certainembodiments, ring Q is a 5-membered non-aromatic ring with 0 hetero-atomin the backbone. In certain embodiments, ring Q is a 5-memberednon-aromatic substituted or unsubstituted cycloalkyl ring.

Structural formula (I_(b)) shows an exemplary embodiment wherein Q is a5-membered non-aromatic ring. R₄ may be any suitable group, for example,each is independently selected from hydrogen, halogen, C₁-C₆ alkyl andC₁-C₆ alkyloxy.

In certain embodiments, R₄ is hydrogen (i.e., unsubstituted ring Q), andthe structure is shown as formula (I)

In some instances, R₃ is an acid-labile protecting group. Examples of R₃groups of interest include, but are not limited to,(4,4′-dimethoxytrityl) (DMT), NT (monomethoxytirtyl), trimethoxy trityl,Pixyl (9-phenylxanthyl) and pivaloyl. (Fisher, et al. 1983 Nucleic AcidsRes. 11, 1589-1599.)

In some instances, R is a group selected from the group consisting ofbenzyl alcohol derivatives (except o-methyl benzyl), alpha-methyl arylalcohols derivatives, naphthalene alcohol derivatives, bi-cyclicaliphatic alcohol derivatives, acylthioalkyl alcohol derivatives andamino acid derivatives. In certain cases, the R is aS-(ethyl)benzothioate.

An acylthioalkyl alcohol derivative includes an acylated thiol groupconnected to an alkyl group (e.g., R—C(═O)S-alkyl-, where R is ahydrocarbyl, an aryl or a heteroaryl). In certain instances, the alkylgroup of the acylthioalkyl alcohol derivative is an ethyl group. As usedhere, the terms “S-ethylthioate” and “acylthioethyl” are usedinterchangeably. In some embodiments, the acylthioalkyl alcoholderivative is an S-ethylthioate derivative. In certain cases, theacylthioalkyl alcohol derivative is S-(ethyl)benzothioate. In certainembodiments, the compound of the present disclosure has the structuralformula (I_(d)) of:

wherein each R₇ is independently selected from hydrogen, halogen, ahydrocarbyl (e.g., C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl) and analkyloxy.

In certain embodiments, the compound of the present disclosure has thestructural formula (I_(e)):

wherein each R₇ is independently selected from hydrogen, halogen, ahydrocarbyl (e.g., C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl) and analkyloxy.

In certain embodiments, the compound of the present disclosure has thestructural formula (I_(f)):

wherein R₈ is an aliphatic group.

In certain embodiments, R (in formula (Ia-c) above) is a derivative ofbenzyl alcohol (except o-methyl benzyl) having the structural formula(II):

wherein R_(a) is hydrogen, or alkyl; each R₁₁ is independently hydrogen,alkyl, alkoxy, alkyl-S—, cyano, methylcyano or halogen; and n is 1, 2,or 3. In some embodiments of Formula (II) when Ra is hydrogen, R₁₁ isnot methyl at the ortho-position.

Exemplary groups from which R may be selected include derivatives ofbenzyl alcohol such as:

wherein each R₆ is independently selected from hydrogen, halogen, cyano,methylcyano, and hydrocarbyl (e.g., C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl).

In particular, exemplary groups from which R can be derived include:

wherein R₉ is selected from hydrogen, halogen, cyano, cyanoethyl, C₁-C₆alkyl, C₁-C₆ alkyloxy, nitro and other electron withdrawing groups, R₇is one or more substituent independently selected from hydrogen,halogen, hydrocarbyl (e.g., C1-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl),alkyloxy and an electron withdrawing group.

In some embodiments, R is described by the structure:

wherein R₇ is one or more substituents each independently selected fromhydrogen, halogen, hydrocarbyl (e.g., C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl), alkyloxy and an electron withdrawing group.

In certain embodiments, R is described by the structure:

wherein R₉ is selected from hydrogen, halogen, cyano, cyanoethyl, C₁-C₆alkyl, C₁-C₆ alkyloxy, nitro and an electron withdrawing group.

Exemplary groups from which R may be derived also include alpha-methylaryl alcohol derivative having the structural formula (III):

wherein X is hydrogen, halogen, cyano or trifluoromethyl; Y is hydrogen,alkyl, haloalkyl or alkoxyalkyl; R_(a) is hydrogen or alkyl. In someembodiments of Formula (III), X and Y are not simultaneously hydrogenand when both R_(a) and X is hydrogen, Y is not methyl. Exemplary Rgroups include

wherein R₉ is halogen, cyano, or trifluoromethyl; and R₁₀ is selectedfrom hydrogen, halogen, C₁-C₆ alkyl, C₁-C₆ alkyloxy, nitro and otherelectron withdrawing groups.

R may also be derived from naphthalene alcohol derivatives having thestructural formula (IV):

wherein R₇ is one or more groups, each R₇ is independently selected fromhydrogen, halogen, cyano, methylcyano, hydrocarbyl (e.g., C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl) and alkyloxy; R₅ is selected from hydrogenand hydrocarbyl (e.g., C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl); andR₁₂ is one or more groups, each R₁₂ is independently selected fromhydrogen and alkoxy. In certain cases, R₇ is a single group and R₁₂ is asingle group.

Exemplary groups from which R may be derived also include:

wherein each R₇ is independently selected from hydrogen, halogen, cyano,methylcyano, C₁-C₆ alkyl and C₁-C₆ alkyloxy; R₅ is independentlyselected from hydrogen and hydrocarbyl (e.g., C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl); and R₁₃ is C₁-C₆ alkyl, R may also be selectedfrom bi-cyclic aliphatic alcohol derivatives having the structuralformula (V) or (VI):

wherein R₁₄ is selected from hydrogen, halogen, C₁-C₆ alkyl and C₁-C₆alkyloxy; R₁₅ and R₁₆ are each independently hydrogen, cyano,cyanomethyl, alkoxy, or halogen, and m is 1 or 2.

Exemplary groups from which R may be derived also include:

wherein R₁₄ is selected from hydrogen, halogen, C₁-C₆ alkyl and C₁-C₆alkyloxy; R₁₅ is hydrogen, halogen or C₁-C₆ alkoxy, R₁₆ is hydrogen,cyano, or halogen.

Additionally, exemplary groups from which R may be selected include:

wherein each R₇ is selected from hydrogen, halogen, cyano, methylcyano,C₁-C₆ alkyl and C₁-C₆ alkyloxy; and R₈ is an aliphatic group.

In some embodiments, R is derived from aS-(2-hydroxylethyl)benzothioate-alcohol having the following structure:

wherein R₇ is hydrogen, halogen, cyano, methylcyano, C₁-C₆ alkyl orC₁-C₆ alkyloxy.

In some embodiments, R is derived from aS-(2-hydroxylethyl)alkylthioate-alcohol having the following structure:

wherein R₈ is an aliphatic group.

Additionally, exemplary groups from which R may be selected include:

Methods

Aspects of the present disclosure include a method of synthesizing apolynucleotide using the subject compounds. In some embodiments, themethod includes: (a) providing a nucleoside residue having anunprotected hydroxyl group; and (b) contacting the nucleoside residuewith a nucleoside monomer (e.g., as described herein) to covalently bondthe nucleoside monomer to the nucleoside residue and produce thepolynucleotide.

In some embodiments, the method further includes exposing thepolynucleotide to an oxidizing agent. Any convenient oxidizing agentsmay be utilized. In some embodiments, the method further includesexposing the nucleic acid to a deprotection agent, e.g., to deprotectthe terminal protecting group (e.g., a 3′ or a 5′-acid labile protectinggroup, as described herein) and produce a free hydroxyl terminal capableof further coupling reactions.

In some embodiments, the method further includes reiterating thecontacting step at least once. The steps of the method may be repeateduntil a polynucleotide of a desired length is obtained. In someinstances, the cycles of polynucleotide synthesis are repeated 200 timesor more, such as 250 times or more, 300 times or more, 400 times ormore, 500 times or more, 600 times or more, 700 times or more, 800 timesor more, 900 times or more, 1000 times or more, or even more, until apolynucleotide (e.g., a DNA) of a desired length and sequence isobtained.

In some embodiments of the method, the nucleoside residue is covalentlybound to a solid support. Any convenient supports may be utilized in thesubject methods. Supports of interest include, but are not limited to,planar surfaces such as arrays, beads, and the like. Suitable solidsupports are in some cases polymeric, and may have a variety of formsand compositions and derive from naturally occurring materials,naturally occurring materials that have been synthetically modified, orsynthetic materials. Examples of suitable support materials include, butare not limited to, polysaccharides such as agarose (e.g., thatavailable commercially as Sepharose®, from Pharmacia) and dextran (e.g.,those available commercially under the tradenames Sephadex® andSephacryl®, also from Pharmacia), polyacrylamides, polystyrenes,polyvinyl alcohols, copolymers of hydroxyethyl methacrylate and methylmethacrylate, silicas, teflons, glasses, and the like. The initialmonomer of the polynucleotide to be synthesized on the substrate surfaceis in some cases bound to a linking moiety which is in turn bound to asurface hydrophilic group, e.g., to a surface hydroxyl moiety present ona silica substrate. In certain embodiments of the method said methodfurther comprises cleaving the nucleic acid from the solid support toproduce a free polynucleotide (e.g., a free nucleic acid).

Any convenient polynucleotide synthesis methods, strategies andchemistries may be adapted for use with the subject compositions andmethods. Polynucleotide synthesis chemistries and methods of interestthat may be adapted for use in the subject methods include, but are notlimited to, phosphoramidite, H-phosphonate, phosphodiester,phosphotriester, and phosphite triester, and those methods and materialsdescribed in S. L. Beaucage et al. (1981) Tetrahedron Lett. 22:1859,U.S. Pat. No. 6,222,030 to Dellinger et al.; U.S. patent applicationPubl'n No. US2002/0058802 A1 to Dellinger et al.; and Seio et al. (2001)Tetrahedron Lett. 42 (49):8657-8660.

In certain embodiments, for 3′-to-5′ synthesis, a support-boundnucleoside residue is provided having the following structure

wherein:

represents the solid support (connected via an optional linker) or asupport-bound polynucleotide chain;

R is hydrogen, protected hydroxyl group, fluoro, an alkoxy,O-ethyleneoxyalkyl (O—CH₂CH₂OR), a protected amino, a protected amido,or protected alkylamino wherein when R is hydrogen, the support-boundnucleoside is a deoxyribonucleoside, as will be present in DNAsynthesis, and when R is a protected hydroxyl group, the support-boundnucleoside is a ribonucleoside, as will be present in RNA synthesis; and

B is a nucleobase or a protected nucleobase, e.g. a purine or pyrimidinebase.

In certain embodiments, the nucleobase may be a conventional purine orpyrimidine base, e.g., adenine (A), thymine (T), cytosine (C), guanine(G) or uracil (U), or a protected form thereof, e.g., wherein the baseis protected with a protecting group such as acetyl, difluoroacetyl,trifluoroacetyl, isobutyryl, benzoyl, or the like. The purine orpyrimidine base may also be an analog of the foregoing; suitable analogsinclude, but are not limited to: 1-methyladenine, 2-methyladenine,N⁶-methyladenine, N⁶-isopentyladenine, 2-methylthio-N⁶-isopentyladenine,N,N-dimethyladenine, 8-bromoadenine, 2-thiocytosine, 3-methylcytosine,5-methylcytosine, 5-ethylcytosine, 4-acetylcytosine, 1-methylguanine,2-methylguanine, 7-methylguanine, 2,2-dimethylguanine, 8-bromoguanine,8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,5-ethyluracil, 5-propyluracil, 5-methoxyuracil, 5-hydroxymethyluracil,5-(carboxyhydroxymethyl)uracil, 5-(methylaminomethyl)uracil,5-(carboxymethylaminomethyl)-uracil, 2-thiouracil,5-methyl-2-thiouracil, 5-(2-bromovinyl)uracil, uracil-5-oxyacetic acid,uracil-5-oxyacetic acid methyl ester, pseudouracil,1-methylpseudouracil, queosine, inosine, 1-methylinosine, hypoxanthine,xanthine, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine and2,6-diaminopurine.

In another aspect, the present disclosure provides a method forsynthesizing a DNA using one or more compounds disclosed herein.

In certain embodiments, the synthesized nucleic acid (e.g., a DNA) has asequence of at least about 150 nucleotides, such as at least about 155nucleotides, at least about 160 nucleotides, at least about 165nucleotides, at least about 170 nucleotides, at least about 175nucleotides, at least about 180 nucleotides, at least about 185nucleotides, at least about 190 nucleotides, at least about 195nucleotides, at least about 200 nucleotides, at least about 205nucleotides, at least about 210 nucleotides, at least about 215nucleotides, at least about 220 nucleotides, at least about 225nucleotides, at least about 230 nucleotides, at least about 240nucleotides, at least about 250 nucleotides, at least about 255nucleotides, at least about 260 nucleotides, at least about 270nucleotides, at least about 280 nucleotides, at least about 300nucleotides. In some instances, any one of the embodiments describedabove, the synthesized nucleic acid (e.g., a DNA) has a sequence havingabout 500 nucleotides or less, such as about 400 nucleotides or less, orabout 300 nucleotides or less. In certain embodiments, the synthesizedDNA has a sequence of at least about 200 nucleotides. In certainembodiments, the synthesized DNA has a sequence of between about 150 andabout 500 nucleotides, such as between about 150 and about 400nucleotides, between about 150 and about 300 nucleotides, or betweenabout 200 and about 300 nucleotides.

In certain embodiments, the synthesized DNA is of a length of about200-mer to about 1,000-mer, (e.g., containing, inter alia, from about200-mer to about 800-mer, from about 200-mer to about 500-mer, fromabout 300-mer to about 800-mer, from about 300-mer to about 500-mer). Incertain embodiments, the synthesized DNA has 2 or fewer singlenucleotide deletions per 100 nucleotides. In certain embodiments, thesynthesized DNA has 1 or less single nucleotide deletions per 100nucleotides.

In some embodiments, the method further includes coupling a first freenucleic acid with a second free nucleic acid to produce an extended freenucleic acid having a length containing, inter alia, from about 300 toabout 10,000 nucleotides. As used herein, the term extended free nucleicacid refers to a free nucleic acids that is produced by fragmentcondensation of nucleic acid fragments synthesized using the subjectlinear stepwise synthesis methods. Any convenient nucleic acid fragmentcoupling methods may be utilized to assembly larger extended nucleicacid molecules from nucleic acids fragments of interest produced by thesubject linear stepwise synthesis methods. In certain embodiments, themethod further includes coupling (e.g., fragment condensation) of one ormore additional free nucleic acids to the extended free nucleic acid toproduce a gene.

Aspects of the present disclosure further include the nucleic acidproducts of the subject methods. The nucleic acid products, e.g., RNA,DNA, of the methods of the disclosure may vary in size, ranging incertain embodiments from 200 or more monomeric units in length, such as250 or more, 300 or more, 350 or more, 400 or more, 450 or more, 500 ormore, or even more. In some embodiments, the nucleic acid products are200 to 1000 monomeric units in length, including, inter alia, 200 to 500monomeric units in length, such as 200 to 400 or 300 to 500 monomericunits in length, In certain embodiments, the nucleic acid product has 1or less (e.g., 1 in 150 nucleotides) single nucleotide deletions per 100nucleotides and no multiple nucleotide deletions.

As stated before, the synthetic methods of the present disclosure may beconducted on a solid support having a surface to which chemical entitiesmay bind. In some embodiments, multiple oligonucleotides beingsynthesized are attached, directly or indirectly, to the same solidsupport and may form part of an array. An “array” is a collection ofseparate molecules of known monomeric sequence each arranged in aspatially defined and a physically addressable manner, such that thelocation of each sequence is known. The number of molecules, or“features,” that can be contained on an array will largely be determinedby the surface area of the substrate, the size of a feature and thespacing between features, wherein the array surface may or may notcomprise a local background region represented by non-feature area.Arrays can have densities of up to several hundred thousand or morefeatures per cm², such as 2,500 to 200,000 features/cm². The featuresmay or may not be covalently bonded to the substrate. An “array,” or“chemical array’ used interchangeably includes any one-dimensional,two-dimensional or substantially two-dimensional (as well as athree-dimensional) arrangement of addressable regions bearing aparticular chemical moiety or moieties (such as ligands, e.g.,biopolymers such as polynucleotide or oligonucleotide sequences (nucleicacids), polypeptides (e.g., proteins), carbohydrates, lipids, etc.)associated with that region. An array is “addressable” when it hasmultiple regions of different moieties (e.g., different polynucleotidesequences) such that a region (i.e., a “feature” or “spot” of the array)at a particular predetermined location (i.e., an “address”) on the arraywill detect a particular target or class of targets (although a featuremay incidentally detect non-targets of that feature). Array features aretypically, but need not be, separated by intervening spaces. In the caseof an array, the “target” will be referenced as a moiety in a mobilephase (typically fluid), to be detected by probes (“target probes”)which are bound to the substrate at the various regions. However, eitherof the “target” or “probe” may be the one which is to be evaluated bythe other (thus, either one could be an unknown mixture of analytes,e.g., polynucleotides, to be evaluated by binding with the other).

In some embodiments, the array is part of a microfluidic device, and istwo or three-dimensional. The solid support comprising such an array,may be substantially planar or may comprise a plurality ofmicrostructures, such as wells, channels and microchannels, elevatedcolumns or posts.

In some embodiments, oligonucleotides being synthesized are attached toa bead directly or indirectly. The beads may optionally be placed in anarray of wells or channels. Suitable solid supports may have a varietyof forms and compositions and derive from naturally occurring materials,naturally occurring materials that have been synthetically modified, orsynthetic materials.

Examples of suitable support materials include, but are not limited to,silicas, silicon and silicon oxide (including any materials used insemiconductor fabrication), teflons, glasses, polysaccharides such asagarose (e.g., Sepharose® from Pharmacia) and dextran (e.g., Sephadex®and Sephacryl®, also from Pharmacia), polyacrylamides, polystyrenes,polyvinyl alcohols, copolymers of hydroxyethyl methacrylate and methylmethacrylate, and the like. The initial monomer of the oligonucleotideto be synthesized on the substrate surface is typically bound to alinking moiety which is in turn bound to a surface hydrophilic group,e.g., a surface hydroxyl moiety present on a silica substrate. In someembodiments, a universal linker is used. In some other embodiments, theinitial monomer is reacted directly with, e.g., a surface hydroxylmoiety. Alternatively, oligonucleotides can be synthesized firstaccording to the present disclosure, and attached to a solid substratepost-synthesis by any method known in the art. Thus, the presentdisclosure can be used to prepare arrays of oligonucleotides wherein theoligonucleotides are either synthesized on the array, or attached to thearray substrate post-synthesis.

With the efficiency and ease of the present method, oligonucleotidesynthesis can be performed in small or large scales. The quantity ofoligonucleotide made in one complete run of the present method (in onecontainer) can thus be less than a microgram, or in micrograms, tens ofmicrograms, hundreds of micrograms, grams, tens of grams, hundreds ofgrams, or even kilograms.

In some embodiments, an array of nucleic acids is synthesized by themethod and compositions of the present disclosure. In some embodiments,the nucleic acids are kept attached to the array for their use inarray-based applications (such as for example gene expression,cytogenetics, genotyping, transcripts or exons profiling etc.). In otherembodiments, the nucleic acids are all—or sometime only asubset-released from the solid support to produce a library or librariesof nucleic acids, or pools that can be optionally amplified prior orafter cleavage from the solid support. Pools or libraries of nucleicacids can be used for example as baits for selective target enrichment,or used as probes for in situ hybridization assays (e.g. o-FISH) orother hybridization assays, multiplex site-directed mutagenesis,multiplex genome engineering and accelerated evolution (MAGE), genesknockout with libraries encoding siRNAs, shRNAs, miRNAs, genomeengineering with libraries of nucleic acids encoding CRISPR RNAs and/orCas proteins, or the nucleic acids encoding genes or genes fragments canbe further assembled and ligated in to longer DNA fragments, genesand/or genome. In some embodiments, the assembled nucleic acids are DNAhaving a length from about 300 nucleotides to about 10,000 nucleotides.In other embodiments, the length of the assembled nucleic acids may varyin size, ranging in certain embodiments from 300 or more nucleotides inlength, such as 400 or more, 1,000 or more, 2,000 or more, 3,000 ormore, 4,000 or more, 5,000 or more, 6,000 or more, 8,000 or more, 10,000or more, or even more.

Also provided is a library of nucleic acids produced using the subjectcompositions and methods. In some embodiments of the library, thelibrary includes a plurality of nucleic acids, where each nucleic acidis synthesized by a subject method as described herein. Also provided isa library including a plurality of nucleic acids having a length fromabout 300 to about 10,000 nucleotides, wherein each nucleic acid iscomposed of assembled nucleic acid fragments synthesized by a subjectmethod as described herein. The nucleic acids may be free nucleic acids.The plurality of nucleic acids may have sequences that together define agene of interest. The plurality of nucleic acids of the library may beassembled into a gene, e.g., using any convenient methods of fragmentcoupling.

The product nucleic acids find use in a variety of applications,including research, diagnostic and therapeutic applications. Forexample, the product nucleic acids find use in research applicationssuch as genomics, cytogenetics, target enrichment and sequencing,site-directed mutagenesis, synthetic biology, gene synthesis, geneassembly, e.g., as probes, primers, gene fragments, DNA/RNA arrays,libraries of nucleic acids. With respect to diagnostic applications,such as genomics, cytogenetics, oncology, infectious diseases,non-invasive prenatal testing (NIPT), target enrichment and sequencing,the product nucleic acids may also find use as probes (for exampleoligoFISH), primers, gene fragments, transcripts, DNA/RNA arrays,libraries of nucleic acids, libraries of transcripts or other agentsemployed in diagnostic protocols. With respect to therapeuticapplications, the product nucleic acids find use as any DNA, RNA orother nucleic acid therapeutic, such as antisense nucleic acids, in genetherapy applications, gene editing, interfering RNA (i.e., iRNA or RNAi)applications, etc.

EXAMPLES

The following examples illustrate the general synthetic strategy ofcompounds (22-54) described herein.

Synthesis of Various 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-Arylphosphoramidite

Bis(N,N-diisopropylamino) chlorophosphine (Chem Genes, 0.019 mols, 5.20grams) was dissolved in anhydrous dichloromethane (20 mL) in aflame-dried 250-mL Schlenk flask equipped with a magnetic stir bar andseptum under argon. To this solution5′-O-dimethoxytrityl-2′-deoxyribothymidine (Chem Genes, 0.019 mols, 1.0equiv) was added. When the 2′-deoxyribonucleoside was completelydissolved, N,N-Diisopropylethylamine (Sigma Aldrich, 0.057 mol, 3.0equiv) was added and the mixture was allowed to stir under argon at roomtemperature for thirty minutes. The 31P NMR spectrum after thirtyminutes indicated complete conversion of the starting material toproduct. After the solvent was removed in vacuo, the crude product wasisolated by chromatography using a 50-90% gradient of ethyl acetate inhexane containing 1% triethylamine. 31P NMR (CDCl3): δ 115.9 (s).

5′-O-Dimethoxytrityl-2′-deoxyribothymidine 3′-O-phosphorodiamidite (2.58mmols, 2 grams) and the aryl alcohol (1.0 equiv.) were dissolved inanhydrous dichloromethane (15 mL) in a 100 mL round-bottom flask andstirred under argon at room temperature. 1H-Ethylthiotetrazole (0.25 Min anhydrous acetonitrile, Glen Research, VA, 1.0 equiv.) was addeddropwise to the mixture via a syringe over a period of 15 min and themixture was allowed to stir under nitrogen at room temperature until TLCanalysis showed the complete conversion of the starting material to anhigher running spot. The solvent was removed in vacuo, the crude productwas isolated by chromatography using a 50-90% gradient of ethyl acetatein hexane containing 1% triethylamine.

TABLE 1 Phosphorus protecting groups partially removable duringoxidation step (e.g., protecting group R of Formula (I)) Naphtalenemotif

24 (31P NMR: δ 148.7, 147.9)

32 (31P NMR: δ 148.3, 148.0)

33 (31P NMR: δ 148.5, 147.9)

23 (31P NMR: δ 148.5, 148.4)

25 (31P NMR: δ 148.8, 147.9)

34 31P NMR: δ 148.3, 148.1

35 31P NMR: δ 148.6, 148.2 Alpha methyl motif

36 (31P NMR: δ 146.9, 146.4, 146.0)

22 (31P NMR: δ 146.9, 146.4, 146.0)

37 (31P NMR: δ 146.6, 146.3, 145.9)

38 (31P NMR: δ 147.1, 146.5, 146.0)

39 (31P NMR: δ 147.0, 146.9, 146.2, 146.0) Benzyl motif

40 (31P NMR: δ 147.9)

27 (31P NMR: δ 148.9, 148.5)

28 (31P NMR: δ 149.1, 148.6)

41 (31P NMR: δ 148.0, 147.6)

42 (31P NMR: δ 148.0, 147.9)

43 (31P NMR: δ 148.0, 147.5)

44 (31P NMR: δ 148.04, 148.00)

45 (31P NMR: δ 148.5)

46 (31P NMR: δ 148.2, 147.7)

29 (31P NMR: δ 148.23, 148.20) Bicyclic aliphatic motif Fused rings

26 (31P NMR: δ 148.7, 148.2, 148.0, 147.8)

47 (31P NMR: δ 149.1, 149.0, 148.9, 148.8)

48 (31P NMR: δ 148.3, 148.1, 147.8, 147.2)

49 (31P NMR: δ 148.6, 148.2, 147.7, 147.3)

50 (31P NMR: δ 148.2, 147.5, 147.4, 146.8)

51 (31P NMR: δ 148.1, 147.4, 147.82)

52 (31P NMR: δ 148.4, 147.9, 147.0, 146.9)

53 (31P NMR: δ 149.2, 148.5)

TABLE 2 Phosphorus protecting groups removable during ammonia cleavage(e.g., protecting group R of Formula (I)) Acetyl-L-threonine methylester (30)

30 (31P NMR: δ 148.4, 147.0) Acetyl-L-Serine methyl ester (54)

54 S-ethyl benzothioate (31)

31 (31P NMR: δ 148.2, 147.6)

Synthesis of Various 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O—S-(Ethyl)Benzothioate-Pyrrolidinylphosphoramidite

Tris(1-pyrrolidinyl)phosphine (0.016 mols, 4 grams) was dissolved inanhydrous dichloromethane (20 mL) in a 100 mL round-bottom flask andstirred under argon at room temperature. To this solution5′-O-dimethoxytrityl-2′-deoxyribothymidine (Chem Genes, 0.016 mols, 1.0equiv.) was added. In the end 1H-Ethylthiotetrazole (0.25 M in anhydrousacetonitrile, Glen Research, VA, 1.0 equiv) was added dropwise to themixture via a syringe over a period of 15 min and the mixture wasallowed to stir under nitrogen at room temperature for thirty minuteswhen the 31P NMR spectrum indicated complete conversion of the startingmaterial to product. The solvent was removed in vacuo, the crude productwas isolated by chromatography using a 30-60% gradient of ethyl acetatein dichloromethane containing 1% triethylamine. 31P NMR (CDCl3): δ 135.2(s).

5′-O-Dimethoxytrityl-2′-deoxyribothymidine3′-O-bis(1-pyrrolidinyl)phosphorodiamidite (2.80 mmols, 2 grams) and thethioester (1.0 equiv) were dissolved in anhydrous dichloromethane (15mL) in a 100 mL round-bottom flask and stirred under argon at roomtemperature. 1H-Ethylthiotetrazole (0.25 M in anhydrous acetonitrile,Glen Research, VA, 1.0 equiv) was added dropwise to the mixture via asyringe over a period of 15 min and the mixture was allowed to stirunder nitrogen at room temperature until TLC analysis showed thecomplete conversion of the starting material to a higher running spot.The solvent was removed in vacuo, the crude product was isolated bychromatography using a 30-60% gradient of ethyl acetate indichloromethane containing 1% triethylamine. 31P NMR (CDCl3): δ 144.1,144.3 (d)

General Procedure for Solid-Phase Oligonucleotide Synthesis

Synthesis is performed on 0.2 (60mer) and 1.0 micromole (20mer) scaleusing dT-CPG columns (500 Angstrom for 20mer and 1000 Angstrom for60mer) from Glen Research according to the standard DNA cycle on an ABImodel 394 automated DNA synthesizer. The synthetic protocol was based onthe conventional DMT phosphoramidite method. All protecteddeoxyribothymidine 3′-O-arylphosphoramidite were dissolved in anhydrousacetonitrile at a concentration of 100 mM and placed on the appropriateports of the synthesizer. Prior to synthesis, the 5′-DMT group on thesupport-bound 2′-deoxyribonucleoside was removed with 3% dichloroaceticacid in dichloromethane. The activator was 5-ethylthio-1H-tetrazole(0.25 M in anhydrous acetonitrile, Glen Research, VA). After eachcondensation step, the support was washed with acetonitrile for 40 s andUnicap phosphoramidite (Glen Research, VA) was used according to themanufacturer's recommendations for capping failure sequences. Thestandard oxidation reagent (0.02 M iodine in THF/water/pyridine) wasused for oxidation. Postsynthesis oligomers were cleaved from solidsupport by treatment with Ammonia (Macron, solution with 10-35%Ammonia). In several cases, before the cleavage, the oligonucleotidestill joined to CPG was treated with 1 M solution of2-carbamoyl-2-cyanoethylene-1,1-dithiolate in DMF (1 mL) for 4 to 8hours. The resin was then washed with DMF followed by MeOH and driedunder argon and finally cleaved with ammonia. The cleavage mixture wasdiscarded and the solid-support was evaporated to dryness in a SpeedVac.The ODN product adsorbed on the support was dissolved in water andanalyzed by LC-MS.

LC-MS Analysis of Crude Oligonucleotides

Analysis of 20mers and 60mers were performed on Agilent 6530Accurate-Mass Q-TOF LC/MS in negative mode. Data were processed withAgilent Mass Hunter Qualitative Analysis B 04.00 software.

Two different reverse phase columns were used: ACQUITY UPLC BEH C18, 1.7μm, 2.1×100 nm Column for the analysis of 20mers and ACQUITY UPLC PrSTC4, 1.7 μm, 2.1 mm×150 mm, Column for the analysis of 60mers. More thanone eluent system and several gradients were used:

Eluent System 1

Buffer A: 200 mmHFIP, 8 mMTEA, 5% Methanol in waterBuffer B: 90% Methanol in water

Eluent System 2

Buffer A: 5 mM Dibutylammonium Acetate, 5% Organic component in waterBuffer B: 5 mM Dibutylammonium Acetate, 10% water in Organic component

Organic Component: 1:1 Acetonitrile: 2-Isopropanol

TABLE 3 Gradient 1 Time (min) % Buffer B Flow (mL/min) 0.0 5.0 0.2 3.05.0 0.2 30.0 50.0 0.2 45.0 80.0 0.2 47.0 5.0 0.2 70.0 5.0 0.2

TABLE 4 Gradient 2 Time (min) % Buffer B Flow (mL/min) 0.0 15.0 0.2 15.025.0 0.2 30.0 45.0 0.2 35.0 60.0 0.2 40.0 15.0 0.2 45.0 15.0 0.2

TABLE 5 Gradient 3 Time (min) % Buffer B Flow (mL/min) 0.0 5.0 0.2 30.025.0 0.2 35.0 60.0 0.2 40.0 10.0 0.2 45.0 5.0 0.2 50.0 5.0 0.2

All the samples were run in negative mode and the following is a list ofall the mass parameters used.

TABLE 6 Source Parameters Source Parameters AJS ESI (seg) AJS Exp MS TOF(Exp) Gas Temp 325 ° C. V Cap 3800 V Fragmentor 175 V Drying Gas  9l/min Nozzle 100 V Skimmer 65 V Nebulizer 20 psig Voltage OctopoleRF 750V Sheat Gas Temp 250 ° C. (exp) Vpp Sheat Gas Flow 11 l/min

All the samples were processed with the MassHunter Qualitative Analysissoftware to extract chromatograms from various signal types as Total IonChromatogram (TIC), DAD Chromatogram and Extracted Ion Chromatogram(EIC).

The following examples illustrate the analysis of 20mers and 60mers byusing phosphoramidites (22-31) described herein. In particular eacholigonucleotide has been analyzed by TIC, DAD and EIC Chromatograms.

dT₂₀ Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-α-Methyl-p-Bromobenzylphosphoramidite (22)

TIC, DAD chromatograms and Mass analysis of dT₂₀ using1-(4-Bromophenyl)Ethanol as phosphorus protecting group are shown inFIG. 1 and FIG. 1 (cont'd).

dT₂₀ Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-1-Methoxynaphtalenephosphoramidite (23)

TIC, DAD chromatograms and Mass analysis of dT20 using1-methoxy-2-naphtalenemethanol as phosphorus protecting group are shownin FIG. 2 and FIG. 2 (cont'd).

dT20 Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-Naphtalenephosphoramidite (24)

TIC, DAD chromatograms and mass analysis of dT₂₀ using2-naphtalenemethanol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment are shown in FIG.3, FIG. 3 (cont'd), FIG. 3 (cont'd 2) and FIG. 3 (cont'd 3).

dT₂₀ Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-6-Bromo-2-Naphtalenehosphoramidite (25)

TIC, DAD chromatograms and mass analysis of dT20 using6-Bromo-2-naphtalenemethanol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment are shown in FIG.4, FIG. 4 (cont'd) and FIG. 4 (cont'd 2).

dT₂₀ by Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-1-Indane-5-Carbonitrilephosphoramidite (26)

TIC, DAD chromatograms and Mass analysis of dT₂₀ using1-hydroxyindane-5-carbonitrile as phosphorus protecting group are shownin FIG. 5, FIG. 5 (cont'd) and FIG. 5 (cont'd 2).

dT₂₀ Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-4-Cyanobenzylphosphoramidite (27)

TIC, DAD chromatograms and mass analysis of dT₂₀ using 4-cyanobenzylalcohol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment are shown in FIG.6, FIG. 6 (cont'd) and FIG. 6 (cont'd 2).

dT₂₀ Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-3-Cyanobenzylphosphoramidite (28)

TIC, DAD chromatograms and mass analysis of dT₂₀ using 3-cyanobenzylalcohol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment are shown in FIG.7, FIG. 7 (cont'd) and FIG. 7 (cont'd 2).

dT₂₀ by Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-2-Ethynylbenzylphosphoramidite (29)

TIC, DAD chromatograms and mass analysis of dT₂₀ using 2-ethynylbenzylalcohol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment are shown in FIG.8, FIG. 8 (cont'd) and FIG. 8 (cont'd 2).

dT₂₀ Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-Acetyl-L-Threoninemethylesterphosphoramidite (30)

TIC, DAD chromatograms and Mass analysis of dT₂₀ usingacetyl-L-threoninemethylester as phosphorus protecting group are shownin FIG. 9, FIG. 9 (cont'd), FIG. 9 (cont'd 2), FIG. 9 (cont'd 3) andFIG. 9 (cont'd 4).

dT₂₀ using 5′-O-Dimethoxytrityl-2′-deoxyribothymidine3′-O—S-ethylbenzothioate-pyrrolidinylphosphoramidite (31)

DAD chromatogram and Mass analysis of dT₂₀ using S-ethylbenzothioate asphosphorus protecting group are shown in FIG. 10 and FIG. 10 (cont'd).

dT₆₀ Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O-4-Cyanobenzylphosphoramidite (27) (27)

TIC, DAD chromatograms and mass analysis of dT₂₀ using 4-cyanobenzylalcohol as phosphorus protecting group with a2-carbamoyl-2-cyanoethylene-1,1-dithiolate treatment are shown in FIG.11, FIG. 11 (cont'd) and FIG. 11 (cont'd 2).

dT₆₀ Using 5′-O-Dimethoxytrityl-2-Deoxyribothymidine3′-O-Acetyl-L-Threoninemethylesterphosphoramidite (30)

TIC, DAD chromatograms and Mass analysis of dT₆₀ usingAcetyl-L-threoninemethylester as phosphorus protecting group are shownin FIG. 12, FIG. 12 (cont'd) and FIG. 12 (cont'd 2).

dT60 by Using 5′-O-Dimethoxytrityl-2′-Deoxyribothymidine3′-O—S-Ethylbenzothioate-Pyrrolidinylphosphoramidite (31)

TIC, DAD chromatograms and Mass analysis of dT₆₀ usingS-ethylbenzothioate as phosphorus protecting group are shown in FIG. 13,FIG. 13 (cont'd) and FIG. 13 (cont'd 2).

Comparison of a T₆₀ synthesized using S-ethylbenzothioate as phosphorusprotecting group with a T₆₀ synthesized with a standard 2-cyanoethylphosphoramidite is shown in FIG. 14 and FIG. 14 (cont'd).

The following examples illustrate the general synthetic strategy ofadditional compounds (1-15) described herein.

Materials and methods:5′-Dimethoxytrityl-N⁶-dimethylaminoacetamidine-2′-deoxyadenosine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditeDMA-dA-CE, adenosine hydrate, guanosine hydrate, cytidine hydrate,dimethoxytrityl chloride were purchased from Chem Genes,5′-Dimethoxytrityl-N⁴-acetyl-2′-deoxycytidine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Ac-dC-CE),5′-Dimethoxytrityl-N²-isobutyryl-2′-deoxyguanosine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditeiBu-dG-CE, dT-CE, dT-CPG column (1 μmol), dC-CPG column (1 μmol), 0.45Methylthio-tetrazole in acetonitrile, CAP Mix A (THF/Pyridine/Ac₂O) andCAP Mix B (16% MeIm in THF), 0.02M I₂ in THF/Pyridine/H2O, 3%Trichloroacetic acid in dichloromethane, dry acetonitrile were purchasedfrom Glen Research. 4-acetylmorpholine, N,N-dimethyl acetamide diethylacetal, N-Methylpyrrolidone, N,N-Diisopropylethylamine, dry pyridine,Dimethyl Sulphate, NaOMe were purchased from Sigma-Aldrich.

DNA synthesis and LCMS studies: DNAs were synthesized in 394 RNA/DNAsynthesizer (Applied Biosystem. 0.2 μmole method). P(III) chemistry ofphosphoramidite method was used to synthesize DNA in standard four stepcycle viz. deprotection, activation and coupling, oxidation and capping.3% Trichloroacetic acid in dichloromethane was used for thedeprotection, 0.45M ethylthio-tetrazole in acetonitrile was used as theactivating agent. 0.02M I₂ in THF/Pyridine was used for oxidation fromamidite to phosphate. THF/Pyridine/Ac₂O and 16% N-Methylimidazole in THEwere used to cap the sequences that fail to couple. Controlled poreglass (CPG) was used as the immobilized phase. Shorter DNA wassynthesized on the CPG of 500 Å pore size in 1 μmole scale while longersequences (>20 bp) were synthesized on CPG of 1000 Å pore size in 0.2μmole scale. The DNAs were cleaved from the solid support by treatingCPG with 28-30% NH₄OH at 55° C. for overnight. However the cleavage anddeprotection efficiency of NH₄OH was studied for the acetamidine andmorpholinoacetamidine protecting groups at variable temperature fromambient temperature to 55° C. and 2 h to overnight.

The cleaved DNA was extracted into H₂O containing 5% acetonitrile andfiltered. The filtered DNA was directly used for LCMS studies. LCMSspectra were recorded in Agilent 6500 Q-TOF LC/MS system. LC/MS wererecorded by using dibutylammonium acetate/isopropyl alcohol(IPA)/acetonitrile (ACN) as the eluent buffer system. The composition ofbuffer was: Buffer A: 5 mM dibutylammoniumacetate+5% IPA:ACN (1:1),Buffer B: 5 mM dibutylammonium acetate+90% IPA:ACN (1:1).Dibutylammonium acetate was selected for the mobile phase to reduce massspectral signal of higher charge states. It helped to extract EICspectra of the DNA of desired mass. Mass spectra of the DNA was analysedby Agilent MassHunter Workstation Qualitative Analysis B.06.00. EICspectra of oligos with desired mass was extracted from the TICchromatogram by the input of exact and calculated mass value of the DNAor adducts.

Synthesis of Precursor Reagents for the Nucleobase Protection.

N,N-Dimethylacetamide Diethyl Acetal:

N,N-Dimethylacetamide Diethyl acetal was synthesized according to theprocedure of McBride, L. J; Kierzek, R.; Beaucage, S. L.; Caruthers, M.H. J. Am. Chem. Soc., 1986, 108, 2040-2048.

2,2-Dimethoxy-1-methylpyrrolidine (Lu, J.; Khdour, O. M.; Armstrong, J.S.; Hecht, S. M. Bioorg. Med. Chem., 2010, 18, 7628-7638):

A mixture N-methyl-2-pyrrolidinone (26 mL, 273 mmol) and of dimethylsulfate (26 mL, 278 mmol) was stirred and heated at 90° C. for 90 min,then allowed to cool to room temperature. A solution containing 65 mL of25% methanolic sodium methoxide and 135 mL of methanol was added at ˜10°C. under argon over a period of 1 h. The precipitated white solid wasfiltered and the solvent was concentrated under reduced pressure. Theyellow oily residue was dissolved in 250 mL of diethylether and stirredfor another 1 h, then the precipitated solid was filtered again. Thesolid was washed with diethylether. After the ether was concentrated,the residue was distilled in vacuo to give the compound as a pale yellowliquid: yield 14.8 g (37%). ¹H NMR in CHCl₃ is according to theliterature.

Synthesis of 4-(1,1-dimethoxyethyl)morpholine (Feng R., -L; Gong, P.;Fang, L.; Hong, W. Chem. Res. Chinese U, 2005, 21, 177-192):

A mixture 4-acetylmorpholine (32 mL, 278 mmol) and of dimethyl sulfate(26 mL, 278 mmol) was stirred and heated at 90° C. for 60 min, thenallowed to cool to room temperature. A solution containing 65 mL of 25%methanolic sodium methoxide and 135 mL of methanol was added at ˜10° C.under argon over a period of 2 h. The precipitated white solid wasfiltered and the solvent was concentrated under reduced pressure. Theyellow oily residue was dissolved in 250 mL of diethylether and stirredfor another 1 h, then the precipitated solid was filtered again. Thesolid was washed with diethylether. After the ether was concentrated,the residue was used for the next step of the reaction. The weight ofthe crude oil was 22 g.

Synthesis of dA, dC, dG, dT3′-(2-Cynoethyl)-N,N-Diisopropylphosphoramidites

Scheme 1 depicts the synthesis of compound (3) under the followingconditions: (i) 4-(1,1-dimethoxyethyl)morpholine, RT, Overnight; (ii)4,4-Dimethoxytritylchloride, pyridine, N,N-Diisopropylethylamine (2 h);(iii) N,N-Diisopropyl-Cyanoethyl phosphoramiditechloride,N,N-Diisopropylethylamine, CH₂C₂ (2 h).

6-N-(1-(morpholino)ethylidene)-2′-deoxyadenosine (1)

2.69 g (10 mmol) of 2′-Deoxyadenosine monohydrate was co-evaporated withpyridine 3 times and was suspended in 20 mL of dry methanol. 10 g of4-(1,1-dimethoxyethyl)morpholine was added slowly with constant stirringunder the argon. The reaction was allowed to continue for overnight.After the reaction methanol was removed by rotary evaporation. Thesticky residue was washed several times with diethyl ether to obtainnon-sticky white powder. The compound was further purified by silica gelcolumn chromatography with CHCl₃ as the eluent. Methanol (0-10%) wasused as the gradient. After removing the solvent the pure compound (1)appeared as white foam. Yield: 3.19 g (88%), R_(f) (CHCl₃/MeOH 10/2v/v): 0.43, ¹H NMR (CDCl₃, 400 MHz): δ8.22 (s, 1H), 7.54 (s, 1H), 5.77(t, J=6.11 Hz, 1H), 4.03 (d, J=6.4 Hz, 1H), 3.88 (m, 2H), 3.64 (m, 1H),3.37 (m, 4H. O(CH₂)₂), 2.83 (m, 4H, N(CH₂)₂), 2.51-2.29 (m, 2H), 1.73(s, 3H, C—CH₃). ¹³C NMR (CDCl₃, 400 MHz): δ160.85, 154.22, 152.01,147.94, 138.21, 122.11, 88.22, 83.19, 71.53, 68.12, 62.41, 46.36, 40.11,22.38.

5′-O-(Di-p-methoxytrityl)-6-N-(1-(morpholino)ethylidene)-2′-deoxyadenosine(2)

4-N-(1-(Dimethylamino)ethylidene)-2′-deoxyadenosine (1) (3.19 g 0.0088mole) was dissolved in dry pyridine (25 mL) and the solution madesaturated with argon. N,N-diisopropylethylamine (6.42 g, 0.05 mole, 8.65mL) was added to the solution and stirred for 10 min under the argon.Solution of Di-p-methoxytritylchloride (2.95 g, 0.0088 mole) in 25 mL ofdry pyridine was prepared under the argon. The DMT-Cl solution wastransferred to the solution of 1 with the continuous flow of argon. Thereaction was allowed to continue for 2 h with constant stirring underthe argon. Solution was concentrated and further diluted with 100 mL ofdichloromethane. The mixture was washed with saturated aqueous solutionof NaHCO₃ (100 mL) thrice. The organic phase was further washed withsaturated aqueous solution of NaCl (100 mL). The organic phase was driedover anhydrous Na₂SO₄. The compound was further purified by silica gelcolumn chromatography in CH₂Cl₂ (MeOH was used as the gradient, 0-2%)with 3% triethylamine. After removing the solvent, the compound (2)appeared as the white foam. Yield: 4.54 g (78%); Rf (CH₂Cl₂/MeOH 10/0.5v/v): 0.41; ¹H NMR (CDCl₃, 400 MHz): δ8.48 (s, 1H), 7.76 (s, 1H),7.3-6.6 (m, 13H, aryl), 5.84 (t, J=6.31 Hz, 1H), 4.13 (d, J=7.3 Hz, 1H),3.93 (m, 2H), 3.76 (s, 6H, (O—CH₃)₂), 3.54 (m, 1H), 3.33 (m, 4H.O(CH₂)₂), 2.91 (m, 4H, N(CH₂)₂), 2.55-2.31 (m, 2H), 1.61 (s, 3H, C—CH₃).¹³C NMR (CDCl₃, 400 MHz): δ167.15, 157.62, 154.41, 150.37, 147.54,140.22, 140.14, 133.97, 130.21, 128.33, 124.32, 122.38, 112.76, 91.22,86.92, 80.27, 74.22, 67.21, 62.12, 59.37, 47.28, 41.19, 23.3.

5′-O-(Di-p-methoxytrityl)-6-N-(1-(morpholino)ethylidene)-2′-deoxyadenosine-2-cyanoethyl-N,N-diisopropylphosphoramidite(3)

5′-O-(Di-p-methoxytrityl)-4-N-(1-morpholinoethylideneamino)-2′-deoxyadenosine(2) (1.66 g, 0.0025 mole) was dissolved in 10 mL of dry CH₂Cl₂.N,N-diisopropylethylamine (6 mL) was added to the solution withcontinuous flow of argon and stirring.N,N-Diisopropyl-O-cyanoethylchlorophosphoramidite (590 mg, 0.0025 mole)was added to the solution and the reaction was allowed to continue for 2h under argon. After the reaction was over the solution was poured into30 mL of saturated aq. NaHCO₃ solution and extracted with CH₂Cl₂ (3×10mL). The combined organic phases were dried over Na₂SO₄ and the solventwas evaporated. The resulting oil was purified by column chromatographywith CH₂Cl₂ containing 3% triethylamine to give the final compound (3)as white foam. The compound was further purified by cold hexaneprecipitation from dichloromethane solution. Yield: 1.51 g (71%). Rf(CH₂Cl₂/MeOH 10/0.5 v/v): 0.44, ³¹P NMR (CDCl₃, 400 MHz): δ148.74,147.91 (Diastereoisomers).

Scheme 2 1 depicts the synthesis of compound (9) according to thefollowing conditions: (i) N,N-Dimethylacetamide Diethyl acetal, Ethanol,60° C., 24 h; (ii) 4,4-Dimethoxytritylchloride, pyrideine,N,N-Diisopropylethylamine, RT, 2 h); (iii) N,N-Diisopropyl-Cyanoethylphosphoramiditechloride, N,N-Diisopropylethylamine, CH₂Cl₂ (2 h); (iv)4-(1,1-dimethoxyethyl)morpholine, 60° C., Overnight; (v)4,4-Dimethoxytritylchloride, pyrideine, N,N-Diisopropylethylamine (3 h);(vi) N,N-Diisopropyl-Cyanoethyl phosphoramiditechloride,N,N-Diisopropylethylamine, CH₂Cl₂ (2 h).

2-N-(1-(Dimethylamino)ethylidene)-2′-deoxyguanosine (4)

2-N-(1-(Dimethylamino)ethylidene)-2′-deoxyguanosine was synthesizedaccording to the literature procedure with minor modification (Lu, J.;Khdour, O. M.; Armstrong, J. S.; Hecht, S. M. Bioorg. Med. Chem., 2010,18, 7628-7638). 2′-deoxyguanosine dehydrate (3.66 g 12 mmol) wassuspended in dry ethanol. N,N-Dimethylacetamide Diethyl acetal (7.8 g,44 mmol) was added to the suspension with constant stirring. The mixturewas warmed to ˜70° C. for an hour until the solution became homogenized(pale yellow). Stirring was continued for 24 h. Ethanol was removed byrotary evaporation under reduced pressure. The sticky residue was washedseveral times with diethylether till the residue become non-stickysolid. The compound was further purified by column chromatography usingCHCl₃ as the eluent. Methanol was used as the gradient (0-20%). Thecompound was eluted first. After removing the solvent of the columnfractions, the compound (4) was appeared as the foam. Yield: 1.95 g(48%); R_(f)(CHCl₃/MeOH 10/2 v/v): 0.38; ¹H NMR (CDCl₃, 400 MHz): δ8.02(s, 1H), 6.34 (t, J=6.8 Hz, 1H), 5.84 (m, 1H), 4.75 (s, 1H), 4.14 (d,J=6.6 Hz, 1H), 4.14 (s, 1H), 3.86 (m, 2H), 3.5 (m, 2H), 3.16 (s, 6H,N(CH₃)₂), 2.71-2.19 (m, 2H), 2.21 (s, 3H, C—CH₃). ¹³C NMR (CDCl₃, 400MHz): δ162.25, 158.66, 156.26, 147.26, 138.10, 88.70, 86.26, 71.67,62.61, 40.95, 38.18, 30.29, 17.00; ESI MS: 337.1521 [MH]⁺

5′-O-(Di-p-methoxytrityl)-2-N-(1-(dimethylamino)ethylidene)-2′-deoxyguanosine(5)

2-N-(1-(Dimethylamino)ethylidene)-2′-deoxyguanosine (4) (3.98 g 0.0118mole) was dissolved in dry pyridine (25 mL) and the solution madesaturated with argon. N,N-diisopropylethylamine (6.42 g, 0.05 mole, 8.65mL) was added to the solution and stirred for 10 min under the argon.Solution of Di-p-methoxytritylchloride (4.2 g, 0.012 mole) in 25 mL ofdry pyridine was prepared under the argon. The DMT-Cl solution wastransferred to the solution of (4) under with the continuous flow ofargon. The reaction was allowed to continue for 2 h with constantstirring under the argon. Solution was concentrated and further dilutedwith 100 mL of dichloromethane. The mixture was washed with saturatedaqueous solution of NaHCO₃ (100 mL) thrice. The organic phase wasfurther washed with saturated aqueous solution of NaCl (100 mL). Theorganic phase was dried over anhydrous Na₂SO₄. The compound was furtherpurified by silica gel column chromatography in CH₂C₂(MeOH was used asthe gradient, 0-5%) with 3% triethylamine. After removing the solvent,the compound (5) appeared as the foam. Yield: 5.51 g (73%); Rf(CH₂Cl₂/MeOH 10/0.5 v/v): 0.31; ¹H NMR (CDCl₃, 400 MHz): δ7.71 (s, 1H),7.4-6.8 (m, 13H, aryl), 6.33 (t, J=7 Hz, 1H) 4.59 (m, 1H), 4.11 (m, 1H),3.78 (s, 6H, (O—CH₃)₂), 3.6-3.2 (m, 4H), 3.12 (s, 6H, N(CH₃)₂), 2.55 (m,2H), 2.21 (s, 3H, C—CH₃). ¹³C NMR (CDCl₃, 400 MHz): δ162.27, 158.55,156.39, 147.78, 144.53, 135.86, 130.01, 127.90, 126.91, 119.30, 113.20,86.55, 85.52, 82.97, 72.47, 64.20, 55.26, 40.83, 38.56, 37.87, 30.20,16.85, 7.94; ESI MS: 639.3019 [MH]+

5′-O-(Di-p-methoxytrityl)-2-N-(1-(dimethylamino)ethylidene)-2′-deoxyguanosine-2-Cyanoethyl-N,N-diisopropylphosphoramidite(6)

5′-O-(Di-p-methoxytrityl)-2-N-(1-(dimethylamino)ethylidene)-2′-deoxyguanosine(5) (596 mg, 0.93 mmole) was dissolved in 10 mL of dry CH₂Cl₂.N,N-diisopropylethylamine (1 mL) was added to the solution withcontinuous flow of argon and stirring.N,N-Diisopropyl-O-cyanoethylchlorophosphoramidite (440 mg, 1.86 mmol)was added to the solution and the reaction was allowed to continue for 2h under argon. After the reaction was over the solution was poured into30 mL of saturated aq. NaHCO₃ solution and extracted with CH₂Cl₂ (3×10mL). The combined organic phases were dried over Na₂SO₄ and the solventwas evaporated. The resulting oil was purified by column chromatographywith CH₂Cl₂ containing 3% triethylamine to give the final compound (6)as a light yellow foam. Yield: 0.74 g (94%). Rf (CH₂Cl₂/MeOH 10/0.6v/v): 0.45; ¹H NMR (CDCl₃, 400 MHz): δ11.98 (s, 1H), 7.81 (d, J=8 Hz,1H), 7.48-6.77 (m, 13H, aryl), 6.21 (t, J=6.9 Hz, 1H), 4.83 (m, 1H),4.11 (m, 1H), 3.78 (s, 6H, (O—CH₃)₂), 3.6-3.2 (m, 4H), 3.12 (s, 6H,N(CH₃)₂), 2.55 (m, 2H), 2.21 (s, 3H, C—CH₃). ³¹P NMR (CDCl₃, 400 MHz):δ148.36, 147.86 (Diastereoisomers).

2-N-(1-(morpholino)ethylidene)-2′-deoxyguanosine (7)

2.85 g (10 mmol) of 2′-Deoxyguanosine hydrate was co-evaporated withpyridine 3 times and was suspended in 20 mL of dry methanol. 12 g of4-(1,1-dimethoxyethyl)morpholine was added slowly with constant stirringunder the argon. The reaction was allowed to continue for overnight at60° C. till the solution become homogenous and pale yellow in colour.After the reaction methanol was removed by rotary evaporation. Thesticky residue was washed several times with diethylether to obtainnon-sticky white solid. The compound was further purified by silica gelcolumn chromatography with CHCl₃ as the eluent. Methanol (0-20%) wasused as the gradient. After removing the solvent the pure compound (7)appeared as white foam. Yield: 3.31 g (82%), Rf (CHCl₃/MeOH 10/2 v/v):0.32, ¹H NMR (CDCl₃, 400 MHz): δ7.92 (s, 1H), 6.04 (t, J=5.8 Hz, 1H),5.67 (m, 1H), 4.17 (d, J=5.8 Hz, 1H), 3.84 (m, 2H), 3.52 (m, 2H), 3.43(m, 4H. O(CH₂)₂), 2.96 (m, 4H, N(CH₂)₂), 2.71-2.19 (m, 2H), 2.03 (s, 3H,C—CH₃). ¹³C NMR (CDCl₃, 400 MHz): δ160.15, 157.26, 154.51, 148.34,137.11, 87.85, 84.16, 70.28, 66.22, 45.86, 42.35, 38.38, 29.69, 16.80.

5′-O-(Di-p-methoxytrityl)-2-N-(1-(morpholino)ethylidene)-2′-deoxyguanosine(8)

2-N-(1-(morpholino)ethylidene)-2′-deoxyguanosine (7) (3.31 g 0.0087mole) was dissolved in dry pyridine (25 mL) and the solution madesaturated with argon. N,N-diisopropylethylamine (6.42 g, 0.05 mole, 8.65mL) was added to the solution and stirred for 10 min under the argon.Solution of Di-p-methoxytritylchloride (2.9 g, 0.0087 mole) in 25 mL ofdry pyridine was prepared under the argon. The DMT-Cl solution wastransferred to the solution of (10) with the continuous flow of argon.The reaction was allowed to continue for 3 h with constant stirringunder the argon. The solution was concentrated and further diluted with100 mL of dichloromethane. The mixture was washed with saturated aqueoussolution of NaHCO₃ (100 mL) thrice. The organic phase was further washedwith saturated aqueous solution of NaCl (100 mL). The organic phase wasdried over anhydrous Na₂SO₄. The compound was further purified by silicagel column chromatography in CH₂Cl₂ (MeOH was used as the gradient,0-5%) with 3% triethylamine. After removing the solvent, the compound(8) appeared as the white foam. Yield: 4.89 g (83%); Rf (CH₂Cl₂/MeOH10/0.5 v/v): 0.36; ¹H NMR (CDCl₃, 400 MHz): δ7.78 (s, 1H), 7.3-6.6 (m,13H, aryl), 6.03 (t, J=6.8 Hz, 1H) 4.59 (m, 1H), 3.84 (s, 6H, (O—CH₃)₂),3.7-3.4 (m, 4H), 3.33 (m, 4H, O(CH₂)₂), 3.02 (m, 4H, N(CH₂)₂), 2.55 (m,2H), 2.16 (s, 3H, C—CH₃). ¹³C NMR (CDCl₃, 400 MHz): δ168.22, 160.29,158.89, 156.19, 148.18, 143.23, 137.16, 132.11, 128.10, 127.21, 123.22,120.24, 118.60, 113.28, 89.03, 86.55, 85.12, 82.27, 72.47, 66.90, 56.26,45.83, 37.66, 32.87, 30.20, 16.95.

5′-O-(Di-p-methoxytrityl)-2-N-(1(morpholino)ethylidene)-2′-deoxyguanosine-2-cyanoethyl-N,N-diisopropylphosphoramidite(9)

5′-O-(Di-p-methoxytrityl)-2-N-(1-(morpholino)ethylidene)-2′-deoxyguanosine(8) (1.7 g, 0.0025 mole) was dissolved in 10 mL of dry CH₂Cl₂.N,N-diisopropylethylamine (6 mL) was added to the solution withcontinuous flow of argon and stirring.N,N-Diisopropyl-O-cyanoethylchlorophosphoramidite (590 mg, 0.0025 mole)was added to the solution and the reaction was allowed to continue for 2h under argon. After the reaction was over the solution was poured into30 mL of saturated aq. NaHCO₃ solution and extracted with CH₂Cl₂ (3×10mL). The combined organic phases were dried over Na₂SO₄ and the solventwas evaporated. The resulting oil was purified by column chromatographywith CH₂Cl₂ containing 3% triethylamine to give the final compound aswhite foam. The compound (9) was further purified by cold hexaneprecipitation from dichloromethane solution. Yield: 1.62 g (77%). R_(f)(CH₂Cl₂/MeOH 10/0.7 v/v): 0.49, ³¹P NMR (CDCl₃, 400 MHz): δ147.56,146.86 (Diastereoisomers).

Scheme 3 depicts the synthesis of compound (15) according to thefollowing conditions: (i) 2,2-Dimethoxy-1-methylpyrrolidine, methanol,RT (3 h); (ii) 4,4-Dimethoxytritylchloride, pyridine,N,N-Diisopropylethylamine (2 h); (iii) N,N-Diisopropyl-Cyanoethylphosphoramiditechloride, N,N-Diisopropylethylamine, CH₂Cl₂ (2 h); (iv)4-(1,1-dimethoxyethyl)morpholine, RT, Overnight; (v)4,4-Dimethoxytritylchloride, pyrideine, N,N-Diisopropylethylamine (2 h);(vi) N,N-Diisopropyl-Cyanoethyl phosphoramiditechloride,N,N-Diisopropylethylamine, CH₂Cl₂ (2 h).

Synthesis of 4-N—(N-methylpyrrolidin-2-ylidene)-2′-deoxycytidine (10)

2.5 g (10 mmol) of 2′-Deoxycytidine hydrate (2a) was suspended in 20 mLof dry methanol. 3.8 g (26 mmol) of 2,2-Dimethoxy-1-methylpyrrolidinewas added slowly with constant stirring under the argon. The reactionwas allowed to continue for 3 h till the solution become homogenous andpale yellow in colour. After the reaction methanol was removed by rotaryevaporation. The sticky residue was washed several times withdiethylether to obtain non-sticky solid. The compound was furtherpurified by silica get column chromatography with CH₂Cl₂ as the eluent.Methanol (0-20%) was used as the gradient. After removing the solventthe pure compound appeared as a white foam. Yield: 2.61 g (85%); R_(f)(CH₂Cl₂/MeOH 10/2 v/v): 0.3; ¹H NMR (CDCl₃, 400 MHz): δ7.91 (d, J=7.8Hz, 1H), 6.13 (t, J=7.1 Hz, 1H), 6.03 (d, J=8 Hz, 1H), 4.55 (m, 1H),4.01 (m, 1H), 3.88 (m, 2H), 3.48 (m, 2H), 3.11 (m, 2H), 3.05 (3H), 2.41(m, 2H), 2.06 (m, 2H); ¹³C NMR (CDCl₃, 400 MHz): δ172.20, 169.00,156.74, 141.98, 103.40, 87.53, 70.17, 61.71, 51.71, 40.56, 31.93, 30.53,19.71; ESI MS: 309.1583 [MH]⁺

Synthesis of5′-O-(Di-p-methoxytrityl)-4-N—(N-methylpyrrolidin-2-ylidene)-2′-deoxycytidine(11)

4.2 g (0.0136 mole) of4-N—(N-methylpyrrolidin-2-ylidene)-2′-deoxycytidine (10) was dissolvedin 25 mL of dry pyridine. 9.88 mL of N,N-diisopropylethylamine was addedto the solution under argon and stirred for 10 min. 4.8 g (0.0143 mole)of DMT-Cl dissolved in 25 mL of dry pyridine was added under argon andwith constant stirring. The reaction was allowed to continue for 2 h.The solution was concentrated and further diluted with 100 mL ofdichloromethane. The mixture was washed with saturated aqueous solutionof NaHCO₃ (100 mL) thrice. The organic phase was further washed withsaturated aqueous solution of NaCl (100 mL). The organic phase was driedover anhydrous Na₂SO₄. The compound was further purified by silica gelcolumn chromatography in CH₂Cl₂ (MeOH was used as the gradient, 0-5%)with 3% triethylamine. After removing the solvent, the compound (11)appeared as the pale yellow foam. Yield: 6.48 g (78%); Rf (CH₂Cl₂/MeOH10/0.5 v/v): 0.4; ¹H NMR (CDCl₃, 400 MHz): δ7.90 (d, 1H), 7.44-6.80 (m,13H, aryl), 6.42 (t, J=6.8 Hz, 1H), 5.81 (d, J=8 Hz, 1H), 4.54 (m, 1H),4.15 (m, 1H), 3.80 (s, 6H, (O—CH₃)₂), 3.70-3.40 (m, 4H), 3.15 (t, J=6.8Hz, 2H), 3.03 (s, 3H, N—CH₃), 2.68 (m, 2H), 2.21 (m, 2H). ¹³C NMR(CDCl₃, 400 MHz): δ172.09, 169.02, 158.53, 156.59, 144.54, 140.50,130.10, 128.17, 127.91, 126.91, 113.22, 103.17, 86.64, 86.44, 86.01,71.52, 63.31, 55.24, 51.55, 42.03, 31.82, 30.63, 19.71; ESI MS: 611.2997[MH]⁺

5′-O-(Di-p-methoxytrityl)-4-N—(N-methylpyrrolidin-2-ylidene)-2′-deoxycytidine-2-cyanoethyl-N,N-diisopropylphosphoramidite(12)

5′-O-(Di-p-methoxytrityl)-4-N—(N-methylpyrrolidin-2-ylidene)-2′-deoxycytidine(11) (456 mg, 0.75 mmole) was dissolved in 10 mL of dry CH₂Cl₂.N,N-diisopropylethylamine (1 mL) was added to the solution withcontinuous flow of argon and stirring.N,N-Diisopropyl-O-cyanoethylchlorophosphoramidite (350 mg, 1.48 mmol)was added to the solution and the reaction was allowed to continue for 2h under argon. After the reaction was over the solution was poured into30 mL of saturated aq. NaHCO₃ solution and extracted with CH₂Cl₂ (3×10mL). The combined organic phases were dried over Na₂SO₄ and the solventwas evaporated. The resulting oil was purified by column chromatographywith CH₂Cl₂ containing 3% triethylamineto give the final compound (12)as a light yellow foam. Yield: 0.53 g (87%). R_(f)(CH₂Cl₂/MeOH 10/0.8v/v): 0.5; ³¹P NMR (CDCl₃, 400 MHz): δ149.48, 148.78 (Diastereoisomers)

4-N-(1-(morpholino)ethylidene)-2′-deoxycytidine (13)

2.45 g (10 mmol) of 2′-Deoxycytidine monohydrate was co-evaporated withpyridine 3 times and was suspended in 20 mL of dry methanol. 10 g of4-(1,1-dimethoxyethyl)morpholine was added slowly with constant stirringunder the argon. The reaction was allowed to continue for overnight.After the reaction methanol was removed by rotary evaporation. Thesticky residue was washed several times with diethylether tillnon-sticky white powder was obtained. The compound was further purifiedby silica gel column chromatography with CHCl₃ as the eluent. Methanol(0-10%) was used as the gradient. After removing the solvent the purecompound (13) appeared as white foam. Yield: 3.0 g (88%),R_(f)(CHCl₃/MeOH 10/2 v/v): 0.38; ¹H NMR (CDCl₃, 400 MHz): δ7.53 (d,J=6.3 Hz, 1H), 6.01 (t, J=6.7 Hz, 1H), 5.27 (d, J=7.4 Hz, 1H), 4.35 (m,1H), 3.91 (m, 1H), 3.88 (m, 2H), 3.55 (m, 4H, O(CH₂)₂), 2.91 (m, 4H,N(CH₂)₂), 2.5-2.3 (m, 2H), 1.73 (s, 3H, C—CH₃). ¹³C NMR (CDCl₃, 400MHz): δ163.85, 160.12, 153.81, 142.14, 108.11, 92.17, 84.72, 71.60,67.08, 60.40, 44.78, 40.92, 22.98.

5′-O-(Di-p-methoxytrityl)-4-N-(1-(morpholino)ethylidene)-2′-deoxycytidine(14)

4-N-(1-((morpholino)ethylidene))-2′-deoxycytidine (13) (2.98 g 0.0088mole) was dissolved in dry pyridine (25 mL) and the solution madesaturated with argon. N,N-diisopropylethylamine (6.42 g, 0.05 mole, 8.65mL) was added to the solution and stirred for 10 min under the argon.Solution of Di-p-methoxytritylchloride (2.95 g, 0.0088 mole) in 25 mL ofdry pyridine was prepared under the argon. The DMT-Cl solution wastransferred to the solution of 16 with the continuous flow of argon. Thereaction was allowed to continue for 2 h with constant stirring underthe argon. Solution was concentrated and further diluted with 100 mL ofdichloromethane. The mixture was washed with saturated aqueous solutionof NaHCO₃ (100 mL) thrice. The organic phase was further washed withsaturated aqueous solution of NaCl (100 mL). The organic phase was driedover anhydrous Na₂SO₄. The compound was further purified by silica gelcolumn chromatography in CH₂Cl₂ (MeOH was used as the gradient, 0-2%)with 3% triethylamine. After removing the solvent, the compound (14)appeared as the white foam. Yield: 4.25 g (75%); Rf (CH₂Cl₂/MeOH 10/0.5v/v): 0.38; ¹H NMR (CDCl₃, 400 MHz): δ7.5-6.7 (m, 14H, aryl), 6.05 (t,J=6.7 Hz, 1H), 5.17 (d, J=7.4 Hz, 1H), 4.47 (m, 1H), 4.13 (m, 1H), 3.95(m, 2H), 3.71 (s, 6H, (O—CH₃)₂), 3.48 (m, 4H, O(CH₂)₂), 2.87 (m, 4H,N(CH₂)₂), 2.6-2.3 (m, 2H), 1.67 (s, 3H, C—CH₃); ¹³C NMR (CDCl₃, 400MHz): δ164.75, 161.32, 158.07, 151.71, 147.11, 142.34, 136.21, 129.88,127.62, 125.65, 115.23, 109.11, 92.17, 87.02, 84.72, 71.60, 68.09,61.38, 54.28, 44.71, 41.22, 21.61.

5′-O-(Di-p-methoxytrityl)-4-N-(1-(morpholino)ethylidene)-2′-deoxycytidine-2-cyanoethyl-N,N-diisopropylphosphoramidite(15)

5′-O-(Di-p-methoxytrityl)-4-N-(1-morpholinoethylideneamino)-2′-deoxycytidine(14) (1.60 g, 0.0025 mole) was dissolved in 10 mL of dry CH₂Cl₂.N,N-diisopropylethylamine (6 mL) was added to the solution withcontinuous flow of argon and stirring.N,N-Diisopropyl-O-cyanoethylchlorophosphoramidite (590 mg, 0.0025 mole)was added to the solution and the reaction was allowed to continue for 2h under argon. After the reaction was over the solution was poured into30 mL of saturated aq. NaHCO₃ solution and extracted with CH₂Cl₂ (3×10mL). The combined organic phases were dried over Na₂SO₄ and the solventwas evaporated. The resulting oil was purified by column chromatographywith CH₂Cl₂ containing 3% triethylamine to give the final compound aswhite foam. The compound (15) was further purified by cold hexaneprecipitation from dichloromethane solution. Yield: 1.4 g (66%). R_(f)(CH₂Cl₂/MeOH 10/0.5 v/v): 0.4, ³¹P NMR (CDCl₃, 400 MHz): δ147.39, 146.13(Diastereoisomers).

The following examples illustrate the general synthetic strategy ofcompounds (16-21) describe herein.

Synthesis of5′-O-Dimethoxytrityl-2′-deoxyribonucleoside-3′-O—S-ethylbenzothioate-pyrrolidinylphosphoramidite

Bis(1-pyrrolidinyl)chlorophosphine (0.016 mols, 4 grams) was dissolvedin anhydrous dichloromethane (20 mL) in a 250 mL round-bottom flask andTriethylamine (1 eq) was added to the solution and stirred under argonat room temperature. In the end5′-O-dimethoxytrityl-2′-deoxyribonucleoside (0.016 mols, 1.0 equiv) wasadded to this solution. The mixture was allowed to stir under nitrogenat room temperature for thirty minutes when the 31P NMR spectrumindicated complete conversion of the starting material to product. Atthis point ethyl thiosalicylate (1.0 equiv) was added to the mixture and1H-ethylthiotetrazole (0.25 M in anhydrous acetonitrile, Glen Research,VA, 0.5 equiv) was added immediately after dropwise via a syringe over aperiod of 15 min. The mixture was allowed to stir under nitrogen at roomtemperature until the ³¹P NMR spectrum indicated complete conversion ofthe phosphorodiamidite. The solvent was removed in vacuo, the crudeproduct was isolated by Agilent preparative HPLC SD-1 system using a0-60% gradient of acetonitrile in ethyl acetate containing 2%trimethylamine. The following scheme depicts the synthesis describedabove:

Compound (16):5′-O-Dimethoxytrityl-2′-deoxyribothymidine-3′-O—S-ethylbenzothioate-pyrrolidinylphosphoramidite.³¹P NMR (CD₃CN): δ 143.22, 142.57(d). ESI-MS (m/z): calc. 826.2922(M+H)+, found 826.2921 (M+H)⁺.

Compound (17):5′-O-Dimethoxytrityl-N⁶—(N,N-dimethylamidino)-2′-deoxyriboadenosine-3′-O—S-ethylbenzothioate-pyrrolidinylphosphoramidite.³¹P NMR (CD₃CN): δ 142.92, 142.79(d). ESI-MS (m/z): calc. 904.3616(M+H)⁺, found 904.3607 (M+H)⁺.

Compound (18):5′-O-Dimethoxytrityl-N⁴-acetyl-2′-deoxyribocytidine-3′-O—S-ethylbenzothioate-pyrrolidinylphosphoramidite.³¹P NMR (CD₃CN): δ 143.70, 143.17(d). ESI-MS (m/z): calc. 853.3031(M+H)+, found 853.3035 (M+H)⁺.

Compound (19):5′-O-Dimethoxytrityl-N⁴—(N-methyl-2-pyrrolidinyldene)-2′-deoxyribocytidine-3′-O—S-ethylbenzothioate-pyrrolidinylphosphoramidite.³¹P NMR (CD₃CN): δ 143.41, 142.80(d). ESI-MS (m/z): calc. 892.3504(M+H)⁺, found 853.3506 (M+H)⁺.

Compound (20):5′-O-Dimethoxytrityl-N²-isobutyryl-2′-deoxyriboguanosine-3′-O—S-ethylbenzothioate-pyrrolidinylphosphoramidite.³¹P NMR (CD₃CN): δ 143.17, 143.13(d). ESI-MS (m/z): calc. 921.3406(M+H)⁺, found 921.3385 (M+H)⁺.

Compound (21):5′-O-Dimethoxytrityl-N²—(N,N-dimethylamidino)-2′-deoxyriboguanosine-3′-O—S-ethylbenzothioate-pyrrolidinylphosphoramidite.³¹P NMR (CD₃CN): δ 142.92, 142.79(d). ESI-MS (m/z): calc. 920.3566(M+H)⁺, found 920.3561 (M+H)⁺.

Synthesis of Arrays

A DNA microarray was manufactured on an Agilent array writer accordingto Agilent manufacturing process described by Leproust et al. in NucleicAcids Research (2010), Vol. 38(8), pp 2522-2540. A set of 5,528 uniqueoligonucleotide sequences containing A, G, C, T and ranging from 178 to202 nucleotides long were synthesized with compounds: T(16), C(18),A(17) and G(20). The oligonucleotides were in situ synthesized on acleavable linker on a silylated 6.625×6 in. wafer. At the end of thesynthesis, the oligonucleotides were deprotected and cleaved fromindividual slide by an overnight ammonia gas treatment performed at roomtemperature. The sequences of the oligonucleotides included:

-   -   5′ Adapter=29 nucleotides    -   Sequencing Primer=32 nucleotides    -   Barcode=16 bases    -   Query Sequence=76 or 100 bases    -   3′ Adapter=25 nucleotides        required to perform a sequencing analysis on an Illumina MiSeq        system. The sequence analysis confirmed the integrity and the        presence of the full length sequences. Additionally, a single        200-mer oligonucleotide sequence        5′-AATGATACGGCGACCACCGAGATCTACACCGACAGGTTCAGAGTTCTACAGTCCGA        CGATCGACGTTCCCAGGATACTTATAGGAGGGGCAAACCTCTTCTCTAGAGTCGCTG        GTCCTATCCAGTAAACCACTTGGTTAATGTAAGAGGCCCGCCTTTCGATCAGAAACG        TCTGGATCTCGTATGCCGTCTTCTGCTTGT-3′ (SEQ ID NO: 1) was synthesized        on an array using the same protocol and the same phosphoramidite        compounds: T(16), A(17), C(18) and G(20). The deprotection and        cleavage from the array was performed as described before.        Denaturing polyacrylamide gel electrophoresis of the product        confirmed the successful synthesis of the 200mer (FIG. 25).

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples disclosed herein are intended to helpillustrate the invention, and are not intended to, nor should they beconstrued to, limit the scope of the invention. Indeed, variousmodifications of the invention and many further embodiments thereof, inaddition to those shown and described herein, will become apparent tothose skilled in the art from the full contents of this document,including the examples which follow and the references to the scientificand patent literature cited herein. The examples described hereincontain important additional information, exemplification and guidancethat can be adapted to the practice of this invention in its variousembodiments and equivalents thereof.

Embodiments

Aspects of the present disclosure include a compound having thestructural formula (I):

wherein B is a nucleobase or an analogue thereof, each of R₁ and R₂ isindependently a linear, branched or cyclic, substituted orun-substituted alkyl, or R₁ and R₂ together form a 5-, 6-, 7- or8-membered non-aromatic ring; R₃ is an acid-labile protecting group; andR is a group selected from the group consisting of benzyl alcoholderivatives, alpha-methyl aryl alcohol derivatives, naphthalene alcoholderivatives, bi-cyclic aliphatic alcohol derivatives, S-ethylthioate andamino acid derivatives, with the proviso that R is not o-methyl benzyl.

In some embodiments of the compound, B is a nucleobase or a protectednucleobase, wherein the nucleobase is selected from adenine, guanine,thymine, cytosine and uracil; and R₃ is a group selected from DMT, MNT,TMT, pixyl and pivaloyl. In some embodiments of the compound, each of R₁and R₂ is independently is a linear, branched or cyclic, substituted orun-substituted C₁-C₁₈ alkyl. In some embodiments of the compound, eachof R₁ and R₂ independently is a linear or branched un-substituted C₁-C₆alkyl. In some embodiments, the compound has the structural formula(I_(a)):

In some embodiments of the compound, R₁ and R₂ together form a 5- or6-membered non-aromatic ring, wherein the ring has 0 or 1 hetero-atom inthe backbone. In some embodiments of the compound, R₁ and R₂ togetherform a 5-membered non-aromatic ring with 0 hetero-atoms in the backbone.In some embodiments, the compound has the structural formula (I_(b)):

wherein each R₄ is selected from hydrogen, halogen, C₁-C₆ alkyl andC₁-C₆ alkyloxy. In some embodiments, the compound has the structure offormula I_(c)

In some embodiments, the compound has the structural formula (I_(d)):

wherein each R₇ is independently hydrogen, halogen, C₁-C₆ alkyl or C₁-C₆alkyloxy.

In some embodiments, the compound has the structural formula (I_(f)):

wherein R₈ is an aliphatic group.

In some embodiments of the compound, R is a derivative of benzyl alcoholhaving the structural formula (II):

wherein: R_(a) is hydrogen or alkyl; each R₁₁ is independently hydrogen,alkyl, alkoxy, alkyl-S—, cyano, methylcyano or halogen; and n is 1, 2 or3.

In some embodiments of the compound, R is a derivative of benzyl alcoholselected from:

wherein each R₆ is independently selected from hydrogen, halogen, cyano,methylcyano and hydrocarbyl. In some embodiments of the compound, R is aderivative of benzyl alcohol having the structure:

wherein R₇ is one or more substituents each independently selected fromhydrogen, halogen, C₁-C₆ alkyl, C₁-C₆ alkyloxy and an electronwithdrawing group.

In some embodiments of the compound, R is a derivative of analpha-methyl aryl alcohol having the structural formula (III):

wherein X is hydrogen, halogen, cyano, methylcyano or trifluoromethyl; Yis hydrogen, alkyl, haloalkyl or alkoxyalkyl; and R_(a) is hydrogen oralkyl.

In some embodiments of the compound, R is a derivative of analpha-methyl aryl alcohol selected from:

wherein R₉ is halogen, cyano or trifluoromethyl; and R₁₀ is selectedfrom hydrogen, halogen, C₁-C₆ alkyl and C₁-C₆ alkyloxy.

In some embodiments of the compound, R is a derivative of a naphthalenealcohol having the structural formula (IV):

wherein R₇ is selected from hydrogen, halogen, C₁-C₆ alkyl and C₁-C₆alkyloxy; R₅ is selected from hydrogen and hydrocarbyl; and R₁₂ isselected from hydrogen and alkoxy.

In some embodiments of the compound, R is a derivative of a naphthalenealcohol selected from:

wherein R₁₃ is C₁-C₆ alkyl.

In some embodiments of the compound, R is a derivative of a bi-cyclicaliphatic alcohol having the structural formula (V) or (VI):

wherein R₁₄ is selected from hydrogen, halogen, C₁-C₆ alkyl and C₁-C₆alkyloxy; R₁₅ and R₁₆ are each independently hydrogen, cyano, alkoxy, orhalogen; and m is 1 or 2.

In some embodiments of the compound, R is a derivative of a bi-cyclicaliphatic alcohol selected from:

wherein R₁₄ is hydrogen, halogen, C₁-C₆ alkyl or C₁-C₆ alkyloxy; R₁₅ ishydrogen, halogen or C₁-C₆ alkoxy; and R₁₆ is hydrogen, cyano, orhalogen.

In some embodiments of the compound, R is:

wherein R₇ is selected from hydrogen, halogen, hydrocarbyl and alkyloxy.

In some embodiments of the compound, R is selected from:

In some embodiments of the compound, R is

wherein R₇ is hydrogen, halogen, C₁-C₆ alkyl or C₁-C₆ alkyloxy.

In some embodiments of the compound, R is

wherein R₈ is an aliphatic group.

In some embodiments of the compound, B comprises an amidine nucleobaseprotecting group. In some embodiments of the compound, the amidinenucleobase protecting group is an acetamidine protecting group. In someembodiments of the compound, the acetamidine protecting group isdescribed by the following structure:

wherein R₂₁ and R₂₂ are each independently an alkyl, a substitutedalkyl, or R₂₁ and R₂₂ are cyclically linked to form a 5 or 6 memberedsubstituted or unsubstituted heterocycle. In some embodiments of thecompound, R₂₁ and R₂₂ are methyl. In some embodiments of the compound,R₂₁ and R₂₂ are cyclically linked to form a 5 or 6 membered substitutedor unsubstituted heterocycle, wherein the heterocycle is selected fromthe group consisting of morpholine, piperidine and pyrrolidine.

In some embodiments of the compound, acetamidine protecting group isdescribed by the following structure:

In some embodiments of the compound, B is described by one of thefollowing structures:

In some embodiments of the compound, R is

wherein R₇ is selected from hydrogen, halogen, hydrocarbyl and alkyloxy.

Also provided is a compound having the structural formula (VII)

wherein each of R₁ and R₂ is an isopropyl or R₁ and R₂ together form apyrrolidine heterocyclic ring with N to which they are attached; R₃ isan acid-labile protecting group; R′ is a group selected from acyanoethyl group and a methyl group; and B is a protected nucleobaseselected from:

Also provided is a method of synthesizing a polynucleotide, the methodcomprising: (a) providing a nucleoside residue having an unprotectedhydroxyl group; and (b) contacting the nucleoside residue with anucleoside monomer (e.g., as described herein) to covalently bond thenucleoside monomer to the nucleoside residue and produce thepolynucleotide. In some embodiments, the method further includesexposing the polynucleotide to an oxidizing agent. In some embodiments,the method further includes exposing the nucleic acid to a deprotectionagent. In some embodiments, the method further comprises reiterating thecontacting step at least once. In some embodiments of the method, thenucleoside residue is covalently bound to a solid support. In someembodiments, the method further comprises cleaving the nucleic acid fromsaid solid support to produce a free nucleic acid. In some embodimentsof the method, the nucleic acid is a DNA having a sequence of at leastabout 200 nucleotides. In some embodiments of the method, the nucleicacid is a DNA having a length of about 200 to about 1,000 nucleotides.In some embodiments of the method, the DNA has a length of about 300 toabout 500 nucleotides. In some embodiments of the method, the DNA hasless than 2 single nucleotide deletions per 100 nucleotides. In someembodiments of the method, the DNA has 1 or less single nucleotidedeletions per 100 nucleotides. In some embodiments, the method furtherincludes coupling a first free nucleic acid with a second free nucleicacid to produce an extended free nucleic acid having a length from about300 to about 10,000 nucleotides. In some embodiments, the method furtherincludes coupling one or more additional free nucleic acids to theextended free nucleic acid to produce a gene.

Also provided is a nucleic acid product produced by any one of theembodiments of the subject method described above. Also provided is anarray of nucleic acids synthesized by the any one of the embodiments ofthe subject method described above. Also provided is a library,comprising a plurality of nucleic acids synthesized by any one of theembodiments of the subject method described above. Also provided is alibrary, comprising a plurality of nucleic acids having a length fromabout 300 to about 10,000 nucleotides, wherein each nucleic acid iscomposed of assembled nucleic acid fragments synthesized by any one ofthe embodiments of the subject method described above. In someembodiments of the library, the plurality of nucleic acids are assembledinto a gene.

1.-24. (canceled)
 25. An array of nucleic acids synthesized by a methodcomprising: (a) providing a nucleoside residue having an unprotectedhydroxyl group; and (b) contacting the nucleoside residue with anucleoside monomer to covalently bond the nucleoside monomer to thenucleoside residue, wherein the nucleoside monomer has the structuralformula (I):

wherein B is a nucleobase or an analogue thereof; wherein the Bcomprises an amidine nucleobase protecting group; and wherein theprotecting group is described by the following structure:

wherein R₂₁ and R₂₂ are each independently an alkyl, a substitutedalkyl, or R₂₁ and R₂₂ are cyclically linked to form a 5- or 6-memberedsubstituted or unsubstituted heterocycle; each of R₁ and R₂ isindependently a linear, branched or cyclic, substituted orun-substituted alkyl, or R₁ and R₂ together form a 5-, 6-, 7- or8-membered non-aromatic ring; R₃ is an acid-labile protecting group; andR is a group selected from the group consisting of a benzyl,alpha-methyl aryl, naphthalene, bi-cyclic aliphatic, S-ethylthioate andan amino acid, with the proviso that R is not o-methyl benzyl.
 26. Alibrary comprising a plurality of nucleic acids synthesized by a methodcomprising: (a) providing a nucleoside residue having an unprotectedhydroxyl group; and (b) contacting the nucleoside residue with anucleoside monomer to covalently bond the nucleoside monomer to thenucleoside residue, wherein the nucleoside monomer has the structuralformula (I):

wherein B is a nucleobase or an analogue thereof; wherein the Bcomprises an amidine nucleobase protecting group; and wherein theprotecting group is described by the following structure:

wherein R₂₁ and R₂₂ are each independently an alkyl, a substitutedalkyl, or R₂₁ and R₂₂ are cyclically linked to form a 5- or 6-memberedsubstituted or unsubstituted heterocycle; each of R₁ and R₂ isindependently a linear, branched or cyclic, substituted orun-substituted alkyl, or R₁ and R₂ together form a 5-, 6-, 7- or8-membered non-aromatic ring; R₃ is an acid-labile protecting group; andR is a group selected from the group consisting of a benzyl,alpha-methyl aryl, naphthalene, bi-cyclic aliphatic, S-ethylthioate andan amino acid, with the proviso that R is not o-methyl benzyl.
 27. Thearray of nucleic acids of claim 25, wherein the method further comprisesexposing the nucleic acids to an oxidizing agent.
 28. The array ofnucleic acids of claim 25, wherein the nucleoside residue is covalentlybound to a solid support.
 29. The array of nucleic acids of claim 25,wherein at least some of the nucleic acids are a DNA having a length ofabout 200 to about 1,000 nucleotides.
 30. The library of claim 26,wherein the method further comprises exposing the nucleic acids to anoxidizing agent.
 31. The library of claim 26, wherein the nucleosideresidue is covalently bound to a solid support.
 32. The library of claim26, wherein at least some of the nucleic acids are a DNA having a lengthof about 200 to about 1,000 nucleotides.